Right ventricular preload and afterload challenge induces contractile dysfunction and arrhythmia in isolated hearts of dystrophin-deficient male mice.

Duchenne muscular dystrophy (DMD) is an X-linked recessive myopathy due to mutations in the dystrophin gene. Diaphragmatic weakness in DMD causes hypoventilation and elevated afterload on the right ventricle (RV). Thus, RV dysfunction in DMD develops early in disease progression. Herein, we deliver a 30-min sustained RV preload/afterload challenge to isolated hearts of wild-type (Wt) and dystrophic (Dmdmdx-4Cv) mice at both young (2-6 month) and middle-age (8-12 month) to test the hypothesis that the dystrophic RV is susceptible to dysfunction with elevated load. Young dystrophic hearts exhibited greater pressure development than wild type under baseline (Langendorff) conditions, but following RV challenge exhibited similar contractile function as wild type. Following the RV challenge, young dystrophic hearts had an increased incidence of premature ventricular contractions (PVCs) compared to wild type. Hearts of middle-aged wild-type and dystrophic mice had similar contractile function during baseline conditions. After RV challenge, hearts of middle-aged dystrophic mice had severe RV dysfunction and arrhythmias, including ventricular tachycardia. Following the RV load challenge, dystrophic hearts had greater lactate dehydrogenase (LDH) release than wild-type mice indicative of damage. Our data indicate age-dependent changes in RV function with load in dystrophin deficiency, highlighting the need to avoid sustained RV load to forestall dysfunction and arrhythmia.

PPP1R12C Promotes Atrial Hypocontractility in Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF)-the most common sustained cardiac arrhythmia-increases thromboembolic stroke risk 5-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C (protein phosphatase 1 regulatory subunit 12C)-the PP1 (protein phosphatase 1) regulatory subunit targeting MLC2a (atrial myosin light chain 2)-causes hypophosphorylation of MLC2a and results in atrial hypocontractility. METHODS: Right atrial appendage tissues were isolated from human patients with AF versus sinus rhythm controls. Western blots, coimmunoprecipitation, and phosphorylation studies were performed to examine how the PP1c (PP1 catalytic subunit)-PPP1R12C interaction causes MLC2a dephosphorylation. In vitro studies of pharmacological MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with electrophysiology studies. RESULTS: In human patients with AF, PPP1R12C expression was increased 2-fold versus sinus rhythm controls (P=2.0×10-2; n=12 and 12 in each group) with >40% reduction in MLC2a phosphorylation (P=1.4×10-6; n=12 and 12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF (P=2.9×10-2 and 6.7×10-3, respectively; n=8 and 8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a and dephosphorylation of MLC2a. Mice treated with lentiviral PPP1R12C vector demonstrated a 150% increase in left atrial size versus controls (P=5.0×10-6; n=12, 8, and 12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in mice treated with lentiviral PPP1R12C vector was significantly higher than in controls (P=1.8×10-2 and 4.1×10-2, respectively; n=6, 6, and 5). CONCLUSIONS: Patients with AF exhibit increased levels of PPP1R12C protein compared with controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.

Calcium handling dysfunction and cardiac damage following acute ventricular preload challenge in the dystrophin-deficient mouse heart.

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy and is caused by mutations in the dystrophin gene. Dystrophin deficiency is associated with structural and functional changes of the muscle cell sarcolemma and/or stretch-induced ion channel activation. In this investigation, we use mice with transgenic cardiomyocyte-specific expression of the GCaMP6f Ca2+ indicator to test the hypothesis that dystrophin deficiency leads to cardiomyocyte Ca2+ handling abnormalities following preload challenge. α-MHC-MerCreMer-GCaMP6f transgenic mice were developed on both a wild-type (WT) or dystrophic (Dmdmdx-4Cv) background. Isolated hearts of 3-7-mo male mice were perfused in unloaded Langendorff mode (0 mmHg) and working heart mode (preload = 20 mmHg). Following a 30-min preload challenge, hearts were perfused in unloaded Langendorff mode with 40 µM blebbistatin, and GCaMP6f was imaged using confocal fluorescence microscopy. Incidence of premature ventricular complexes (PVCs) was monitored before and following preload elevation at 20 mmHg. Hearts of both wild-type and dystrophic mice exhibited similar left ventricular contractile function. Following preload challenge, dystrophic hearts exhibited a reduction in GCaMP6f-positive cardiomyocytes and an increase in number of cardiomyocytes exhibiting Ca2+ waves/overload. Incidence of cardiac arrhythmias was low in both wild-type and dystrophic hearts during unloaded Langendorff mode. However, after preload elevation to 20-mmHg hearts of dystrophic mice exhibited an increased incidence of PVCs compared with hearts of wild-type mice. In conclusion, these data indicate susceptibility to preload-induced Ca2+ overload, ventricular damage, and ventricular dysfunction in male Dmdmdx-4Cv hearts. Our data support the hypothesis that cardiomyocyte Ca2+ overload underlies cardiac dysfunction in muscular dystrophy.NEW & NOTEWORTHY The mechanisms of cardiac disease progression in muscular dystrophy are complex and poorly understood. Using a transgenic mouse model with cardiomyocyte-specific expression of the GCaMP6f Ca2+ indicator, the present study provides further support for the Ca2+-overload hypothesis of disease progression and ventricular arrhythmogenesis in muscular dystrophy.

Dystrophic cardiomyopathy: role of the cardiac myofilaments.

Dystrophic cardiomyopathy arises from mutations in the dystrophin gene. Dystrophin forms part of the dystrophin glycoprotein complex and is postulated to act as a membrane stabilizer, protecting the sarcolemma from contraction-induced damage. Duchenne muscular dystrophy (DMD) is the most severe dystrophinopathy, caused by a total absence of dystrophin. Patients with DMD present with progressive skeletal muscle weakness and, because of treatment advances, a cardiac component of the disease (i.e., dystrophic cardiomyopathy) has been unmasked later in disease progression. The role that myofilaments play in dystrophic cardiomyopathy is largely unknown and, as such, this study aimed to address cardiac myofilament function in a mouse model of muscular dystrophy. To assess the effects of DMD on myofilament function, isolated permeabilized cardiomyocytes of wild-type (WT) littermates and Dmdmdx-4cv mice were attached between a force transducer and motor and subjected to contractile assays. Maximal tension and rates of force development (indexed by the rate constant, k tr) were similar between WT and Dmdmdx-4cv cardiac myocyte preparations. Interestingly, Dmdmdx-4cv cardiac myocytes exhibited greater sarcomere length dependence of peak power output compared to WT myocyte preparations. These results suggest dystrophin mitigates length dependence of activation and, in the absence of dystrophin, augmented sarcomere length dependence of myocyte contractility may accelerate ventricular myocyte contraction-induced damage and contribute to dystrophic cardiomyopathy. Next, we assessed if mavacamten, a small molecule modulator of thick filament activation, would mitigate contractile properties observed in Dmdmdx-4cv permeabilized cardiac myocyte preparations. Mavacamten decreased maximal tension and k tr in both WT and Dmdmdx-4cv cardiac myocytes, while also normalizing the length dependence of peak power between WT and Dmdmdx-4cv cardiac myocyte preparations. These results highlight potential benefits of mavacamten (i.e., reduced contractility while maintaining exquisite sarcomere length dependence of power output) as a treatment for dystrophic cardiomyopathy associated with DMD.

Thin filament regulation of cardiac muscle power output: Implications for targets to improve human failing hearts.

The heart's pumping capacity is determined by myofilament power generation. Power is work done per unit time and measured as the product of force and velocity. At a sarcomere level, these contractile properties are linked to the number of attached cross-bridges and their cycling rate, and many signaling pathways modulate one or both factors. We previously showed that power is increased in rodent permeabilized cardiac myocytes following PKA-mediated phosphorylation of myofibrillar proteins. The current study found that that PKA increased power by ∼30% in permeabilized cardiac myocyte preparations (n = 8) from human failing hearts. To address myofilament molecular specificity of PKA effects, mechanical properties were measured in rat permeabilized slow-twitch skeletal muscle fibers before and after exchange of endogenous slow skeletal troponin with recombinant human Tn complex that contains cardiac (c)TnT, cTnC and either wildtype (WT) cTnI or pseudo-phosphorylated cTnI at sites Ser23/24Asp, Tyr26Glu, or the combinatorial Ser23/24Asp and Tyr26Glu. We found that cTnI Ser23/24Asp, Tyr26Glu, and combinatorial Ser23/24Asp and Tyr26Glu were sufficient to increase power by ∼20%. Next, we determined whether pseudo-phosphorylated cTnI at Ser23/24 was sufficient to increase power in cardiac myocytes from human failing hearts. Following cTn exchange that included cTnI Ser23/24Asp, power output increased ∼20% in permeabilized cardiac myocyte preparations (n = 6) from the left ventricle of human failing hearts. These results implicate cTnI N-terminal phosphorylation as a molecular regulator of myocyte power and could serve as a regional target for small molecule therapy to unmask myocyte power reserve capacity in human failing hearts.

Molecular regulation of stretch activation.

Stretch activation is defined as a delayed increase in force after rapid stretches. Although there is considerable evidence for stretch activation in isolated cardiac myofibrillar preparations, few studies have measured mechanisms of stretch activation in mammalian skeletal muscle fibers. We measured stretch activation following rapid step stretches [∼1%-4% sarcomere length (SL)] during submaximal Ca2+ activations of rat permeabilized slow-twitch skeletal muscle fibers before and after protein kinase A (PKA), which phosphorylates slow myosin binding protein-C. PKA significantly increased stretch activation during low (∼25%) Ca2+ activation and accelerated rates of delayed force development (kef) during both low and half-maximal Ca2+ activation. Following the step stretches and subsequent force development, fibers were rapidly shortened to original sarcomere length, which often elicited a shortening-induced transient force overshoot. After PKA, step shortening-induced transient force overshoot increased ∼10-fold following an ∼4% SL shortening during low Ca2+ activation levels. kdf following step shortening also increased after PKA during low and half-maximal Ca2+ activations. We next investigated thin filament regulation of stretch activation. We tested the interplay between cardiac troponin I (cTnI) phosphorylation at the canonical PKA and novel tyrosine kinase sites on stretch activation. Native slow-skeletal Tn complexes were exchanged with recombinant human cTn complex with different human cTnI N-terminal pseudo-phosphorylation molecules: 1) nonphosphorylated wild type (WT), 2) the canonical S22/23D PKA sites, 3) the tyrosine kinase Y26E site, and 4) the combinatorial S22/23D + Y26E cTnI. All three pseudo-phosphorylated cTnIs elicited greater stretch activation than WT. Following stretch activation, a new, elevated stretch-induced steady-state force was reached with pseudo-phosphorylated cTnI. Combinatorial S22/23D + Y26E pseudo-phosphorylated cTnI increased kdf. These results suggest that slow-skeletal myosin binding protein-C (sMyBP-C) phosphorylation modulates stretch activation by a combination of cross-bridge recruitment and faster cycling kinetics, whereas cTnI phosphorylation regulates stretch activation by both redundant and synergistic mechanisms; and, taken together, these sarcomere phosphoproteins offer precision targets for enhanced contractility.

Sarcomeric deficits underlie MYBPC1-associated myopathy with myogenic tremor.

Myosin binding protein-C slow (sMyBP-C) comprises a subfamily of cytoskeletal proteins encoded by MYBPC1 that is expressed in skeletal muscles where it contributes to myosin thick filament stabilization and actomyosin cross-bridge regulation. Recently, our group described the causal association of dominant missense pathogenic variants in MYBPC1 with an early-onset myopathy characterized by generalized muscle weakness, hypotonia, dysmorphia, skeletal deformities, and myogenic tremor, occurring in the absence of neuropathy. To mechanistically interrogate the etiologies of this MYBPC1-associated myopathy in vivo, we generated a knock-in mouse model carrying the E248K pathogenic variant. Using a battery of phenotypic, behavioral, and physiological measurements spanning neonatal to young adult life, we found that heterozygous E248K mice faithfully recapitulated the onset and progression of generalized myopathy, tremor occurrence, and skeletal deformities seen in human carriers. Moreover, using a combination of biochemical, ultrastructural, and contractile assessments at the level of the tissue, cell, and myofilaments, we show that the loss-of-function phenotype observed in mutant muscles is primarily driven by disordered and misaligned sarcomeres containing fragmented and out-of-register internal membranes that result in reduced force production and tremor initiation. Collectively, our findings provide mechanistic insights underscoring the E248K-disease pathogenesis and offer a relevant preclinical model for therapeutic discovery.

Cardiac MyBP-C phosphorylation regulates the Frank-Starling relationship in murine hearts.

The Frank-Starling relationship establishes that elevated end-diastolic volume progressively increases ventricular pressure and stroke volume in healthy hearts. The relationship is modulated by a number of physiological inputs and is often depressed in human heart failure. Emerging evidence suggests that cardiac myosin-binding protein-C (cMyBP-C) contributes to the Frank-Starling relationship. We measured contractile properties at multiple levels of structural organization to determine the role of cMyBP-C and its phosphorylation in regulating (1) the sarcomere length dependence of power in cardiac myofilaments and (2) the Frank-Starling relationship in vivo. We compared transgenic mice expressing wild-type cMyBP-C on the null background, which have ∼50% phosphorylated cMyBP-C (Controls), to transgenic mice lacking cMyBP-C (KO) and to mice expressing cMyBP-C that have serine-273, -282, and -302 mutated to aspartate (cMyBP-C t3SD) or alanine (cMyBP-C t3SA) on the null background to mimic either constitutive PKA phosphorylation or nonphosphorylated cMyBP-C, respectively. We observed a continuum of length dependence of power output in myocyte preparations. Sarcomere length dependence of power progressively increased with a rank ordering of cMyBP-C KO = cMyBP-C t3SA < Control < cMyBP-C t3SD. Length dependence of myofilament power translated, at least in part, to hearts, whereby Frank-Starling relationships were steepest in cMyBP-C t3SD mice. The results support the hypothesis that cMyBP-C and its phosphorylation state tune sarcomere length dependence of myofibrillar power, and these regulatory processes translate across spatial levels of myocardial organization to control beat-to-beat ventricular performance.

Regulation of Myofilament Contractile Function in Human Donor and Failing Hearts.

Heart failure (HF) often includes changes in myocardial contractile function. This study addressed the myofibrillar basis for contractile dysfunction in failing human myocardium. Regulation of contractile properties was measured in cardiac myocyte preparations isolated from frozen, left ventricular mid-wall biopsies of donor (n = 7) and failing human hearts (n = 8). Permeabilized cardiac myocyte preparations were attached between a force transducer and a position motor, and both the Ca2+ dependence and sarcomere length (SL) dependence of force, rate of force, loaded shortening, and power output were measured at 15 ± 1°C. The myocyte preparation size was similar between groups (donor: length 148 ± 10 µm, width 21 ± 2 µm, n = 13; HF: length 131 ± 9 µm, width 23 ± 1 µm, n = 16). The maximal Ca2+-activated isometric force was also similar between groups (donor: 47 ± 4 kN⋅m-2; HF: 44 ± 5 kN⋅m-2), which implicates that previously reported force declines in multi-cellular preparations reflect, at least in part, tissue remodeling. Maximal force development rates were also similar between groups (donor: k tr = 0.60 ± 0.05 s-1; HF: k tr = 0.55 ± 0.04 s-1), and both groups exhibited similar Ca2+ activation dependence of k tr values. Human cardiac myocyte preparations exhibited a Ca2+ activation dependence of loaded shortening and power output. The peak power output normalized to isometric force (PNPO) decreased by ∼12% from maximal Ca2+ to half-maximal Ca2+ activations in both groups. Interestingly, the SL dependence of PNPO was diminished in failing myocyte preparations. During sub-maximal Ca2+ activation, a reduction in SL from ∼2.25 to ∼1.95 µm caused a ∼26% decline in PNPO in donor myocytes but only an ∼11% change in failing myocytes. These results suggest that altered length-dependent regulation of myofilament function impairs ventricular performance in failing human hearts.

Transient receptor potential vanilloid-4 contributes to stretch-induced hypercontractility and time-dependent dysfunction in the aged heart.

AIMS: Cardiovascular disease remains the greatest cause of mortality in Americans over 65. The stretch-activated transient receptor potential vanilloid-4 (TRPV4) ion channel is expressed in cardiomyocytes of the aged heart. This investigation tests the hypothesis that TRPV4 alters Ca2+ handling and cardiac function in response to increased ventricular preload and cardiomyocyte stretch. METHODS AND RESULTS: Left ventricular maximal pressure (PMax) was monitored in isolated working hearts of Aged (24-27 months) mice following preload elevation from 5 to 20mmHg, with and without TRPV4 antagonist HC067047 (HC, 1 µmol/L). In preload responsive hearts, PMax prior to and immediately following preload elevation (i.e. Frank-Starling response) was similar between Aged and Aged+HC. Within 1 min following preload elevation, Aged hearts demonstrated secondary PMax augmentation (Aged>Aged+HC) suggesting a role for stretch-activated TRPV4 in cardiac hypercontractility. However, after 20 min at 20 mmHg Aged exhibited depressed PMax (Aged

Western Diet-Fed, Aortic-Banded Ossabaw Swine: A Preclinical Model of Cardio-Metabolic Heart Failure.

The development of new treatments for heart failure lack animal models that encompass the increasingly heterogeneous disease profile of this patient population. This report provides evidence supporting the hypothesis that Western Diet-fed, aortic-banded Ossabaw swine display an integrated physiological, morphological, and genetic phenotype evocative of cardio-metabolic heart failure. This new preclinical animal model displays a distinctive constellation of findings that are conceivably useful to extending the understanding of how pre-existing cardio-metabolic syndrome can contribute to developing HF.

Regulation of myofilament force and loaded shortening by skeletal myosin binding protein C.

Myosin binding protein C (MyBP-C) is a 125-140-kD protein located in the C-zone of each half-thick filament. It is thought to be an important regulator of contraction, but its precise role is unclear. Here we investigate mechanisms by which skeletal MyBP-C regulates myofilament function using rat permeabilized skeletal muscle fibers. We mount either slow-twitch or fast-twitch skeletal muscle fibers between a force transducer and motor, use Ca2+ to activate a range of forces, and measure contractile properties including transient force overshoot, rate of force development, and loaded sarcomere shortening. The transient force overshoot is greater in slow-twitch than fast-twitch fibers at all Ca2+ activation levels. In slow-twitch fibers, protein kinase A (PKA) treatment (a) augments phosphorylation of slow skeletal MyBP-C (sMyBP-C), (b) doubles the magnitude of the relative transient force overshoot at low Ca2+ activation levels, and (c) increases force development rates at all Ca2+ activation levels. We also investigate the role that phosphorylated and dephosphorylated sMyBP-C plays in loaded sarcomere shortening. We test the hypothesis that MyBP-C acts as a brake to filament sliding within the myofilament lattice by measuring sarcomere shortening as thin filaments traverse into the C-zone during lightly loaded slow-twitch fiber contractions. Before PKA treatment, shortening velocity decelerates as sarcomeres traverse from ∼3.10 to ∼3.00 µm. After PKA treatment, sarcomeres shorten a greater distance and exhibit less deceleration during similar force clamps. After sMyBP-C dephosphorylation, sarcomere length traces display a brief recoil (i.e., "bump") that initiates at ∼3.06 µm during loaded shortening. Interestingly, the timing of the bump shifts with changes in load but manifests at the same sarcomere length. Our results suggest that sMyBP-C and its phosphorylation state regulate sarcomere contraction by a combination of cross-bridge recruitment, modification of cross-bridge cycling kinetics, and alteration of drag forces that originate in the C-zone.

Chronic low-intensity exercise attenuates cardiomyocyte contractile dysfunction and impaired adrenergic responsiveness in aortic-banded mini-swine.

Exercise improves clinical outcomes in patients diagnosed with heart failure with reduced ejection fraction (HFrEF), in part via beneficial effects on cardiomyocyte Ca2+ cycling during excitation-contraction coupling (ECC). However, limited data exist regarding the effects of exercise training on cardiomyocyte function in patients diagnosed with heart failure with preserved ejection fraction (HFpEF). The purpose of this study was to investigate cardiomyocyte Ca2+ handling and contractile function following chronic low-intensity exercise training in aortic-banded miniature swine and test the hypothesis that low-intensity exercise improves cardiomyocyte function in a large animal model of pressure overload. Animals were divided into control (CON), aortic-banded sedentary (AB), and aortic-banded low-intensity trained (AB-LIT) groups. Left ventricular cardiomyocytes were electrically stimulated (0.5 Hz) to assess Ca2+ homeostasis (fura-2-AM) and unloaded shortening during ECC under conditions of baseline pacing and pacing with adrenergic stimulation using dobutamine (1 µM). Cardiomyocytes in AB animals exhibited depressed Ca2+ transient amplitude and cardiomyocyte shortening vs. CON under both conditions. Exercise training attenuated AB-induced decreases in cardiomyocyte Ca2+ transient amplitude but did not prevent impaired shortening vs. CON. With dobutamine, AB-LIT exhibited both Ca2+ transient and shortening amplitude similar to CON. Adrenergic sensitivity, assessed as the time to maximum inotropic response following dobutamine treatment, was depressed in the AB group but normal in AB-LIT animals. Taken together, our data suggest exercise training is beneficial for cardiomyocyte function via the effects on Ca2+ homeostasis and adrenergic sensitivity in a large animal model of pressure overload-induced heart failure. NEW & NOTEWORTHY Conventional treatments have failed to improve the prognosis of heart failure with preserved ejection fraction (HFpEF) patients. Our findings show chronic low-intensity exercise training can prevent cardiomyocyte dysfunction and impaired adrenergic responsiveness in a translational large animal model of chronic pressure overload-induced heart failure with relevance to human HFpEF.

Cardiac myofibrillar contractile properties during the progression from hypertension to decompensated heart failure.

Heart failure arises, in part, from a constellation of changes in cardiac myocytes including remodeling, energetics, Ca2+ handling, and myofibrillar function. However, little is known about the changes in myofibrillar contractile properties during the progression from hypertension to decompensated heart failure. The aim of the present study was to provide a comprehensive assessment of myofibrillar functional properties from health to heart disease. A rodent model of uncontrolled hypertension was used to test the hypothesis that myocytes in compensated hearts exhibit increased force, higher rates of force development, faster loaded shortening, and greater power output; however, with progression to overt heart failure, we predicted marked depression in these contractile properties. We assessed contractile properties in skinned cardiac myocyte preparations from left ventricles of Wistar-Kyoto control rats and spontaneous hypertensive heart failure (SHHF) rats at ~3, ~12, and >20 mo of age to evaluate the time course of myofilament properties associated with normal aging processes compared with myofilaments from rats with a predisposition to heart failure. In control rats, the myofilament contractile properties were virtually unchanged throughout the aging process. Conversely, in SHHF rats, the rate of force development, loaded shortening velocity, and power all increased at ~12 mo and then significantly fell at the >20-mo time point, which coincided with a decrease in left ventricular fractional shortening. Furthermore, these changes occurred independent of changes in ß-myosin heavy chain but were associated with depressed phosphorylation of myofibrillar proteins, and the fall in loaded shortening and peak power output corresponded with the onset of clinical signs of heart failure.NEW & NOTEWORTHY This novel study systematically examined the power-generating capacity of cardiac myofilaments during the progression from hypertension to heart disease. Previously undiscovered changes in myofibrillar power output were found and were associated with alterations in myofilament proteins, providing potential new targets to exploit for improved ventricular pump function in heart failure.

Histone deacetyltransferase inhibitors Trichostatin A and Mocetinostat differentially regulate MMP9, IL-18 and RECK expression, and attenuate Angiotensin II-induced cardiac fibroblast migration and proliferation.

Histone acetylation/deacetylation plays a key role in the epigenetic regulation of multiple pro-fibrotic genes. Here we investigated the effects of histone deacetyltransferase (HDAC) inhibition on angiotensin (Ang)-II-induced pro-fibrotic changes in adult mouse cardiac fibroblasts (CF). CF express class I HDACs 1 and 2, and Ang-II induces their activation. Notably, silencing HDAC1 or HDAC2 attenuated Ang-II induced CF proliferation and migration. Under basal conditions, HDAC1 dimerizes with HDAC2 in CF and Ang-II reversed this interaction. Treatment with Trichostatin A (TSA), a broad-spectrum HDAC inhibitor, restored their physical association, and attenuated Ang-II-induced MMP9 expression, IL-18 induction, and extracellular matrix (collagen I, collagen III and fibronectin) production. Further, TSA inhibited Ang-II-induced MMP9 and Il18 transcription by blocking NF-κB and AP-1 binding to their respective promoter regions. By inhibiting Sp1 binding to RECK promoter, TSA reversed Ang-II-induced RECK suppression, collagen and fibronectin expression, and CF migration and proliferation. The class I-specific HDAC inhibitor Mocetinostat (MGCD) recapitulated TSA effects on Ang-II-treated CF. Together, these results demonstrate that targeting HDACs attenuates the pro-inflammatory and pro-fibrotic effects of Ang-II on CF.

Saxagliptin and Tadalafil Differentially Alter Cyclic Guanosine Monophosphate (cGMP) Signaling and Left Ventricular Function in Aortic-Banded Mini-Swine.

BACKGROUND: Cyclic guanosine monophosphate-protein kinase G-phosphodiesterase 5 signaling may be disturbed in heart failure (HF) with preserved ejection fraction, contributing to cardiac remodeling and dysfunction. The purpose of this study was to manipulate cyclic guanosine monophosphate signaling using the dipeptidyl-peptidase 4 inhibitor saxagliptin and phosphodiesterase 5 inhibitor tadalafil. We hypothesized that preservation of cyclic guanosine monophosphate cGMP signaling would attenuate pathological cardiac remodeling and improve left ventricular (LV) function. METHODS AND RESULTS: We assessed LV hypertrophy and function at the organ and cellular level in aortic-banded pigs. Concentric hypertrophy was equal in all groups, but LV collagen deposition was increased in only HF animals. Prevention of fibrotic remodeling by saxagliptin and tadalafil was correlated with neuropeptide Y plasma levels. Saxagliptin better preserved integrated LV systolic and diastolic function by maintaining normal LV chamber volumes and contractility (end-systolic pressure-volume relationship, preload recruitable SW) while preventing changes to early/late diastolic longitudinal strain rate. Function was similar to the HF group in tadalafil-treated animals including increased LV contractility, reduced chamber volume, and decreased longitudinal, circumferential, and radial mechanics. Saxagliptin and tadalafil prevented a negative cardiomyocyte shortening-frequency relationship observed in HF animals. Saxagliptin increased phosphodiesterase 5 activity while tadalafil increased cyclic guanosine monophosphate levels; however, neither drug increased downstream PKG activity. Early mitochondrial dysfunction, evident as decreased calcium-retention capacity and Complex II-dependent respiratory control, was present in both HF and tadalafil-treated animals. CONCLUSIONS: Both saxagliptin and tadalafil prevented increased LV collagen deposition in a manner related to the attenuation of increased plasma neuropeptide Y levels. Saxagliptin appears superior for treating heart failure with preserved ejection fraction, considering its comprehensive effects on integrated LV systolic and diastolic function.

Molecule specific effects of PKA-mediated phosphorylation on rat isolated heart and cardiac myofibrillar function.

Increased cardiac myocyte contractility by the ß-adrenergic system is an important mechanism to elevate cardiac output to meet hemodynamic demands and this process is depressed in failing hearts. While increased contractility involves augmented myoplasmic calcium transients, the myofilaments also adapt to boost the transduction of the calcium signal. Accordingly, ventricular contractility was found to be tightly correlated with PKA-mediated phosphorylation of two myofibrillar proteins, cardiac myosin binding protein-C (cMyBP-C) and cardiac troponin I (cTnI), implicating these two proteins as important transducers of hemodynamics to the cardiac sarcomere. Consistent with this, we have previously found that phosphorylation of myofilament proteins by PKA (a downstream signaling molecule of the beta-adrenergic system) increased force, slowed force development rates, sped loaded shortening, and increased power output in rat skinned cardiac myocyte preparations. Here, we sought to define molecule-specific mechanisms by which PKA-mediated phosphorylation regulates these contractile properties. Regarding cTnI, the incorporation of thin filaments with unphosphorylated cTnI decreased isometric force production and these changes were reversed by PKA-mediated phosphorylation in skinned cardiac myocytes. Further, incorporation of unphosphorylated cTnI sped rates of force development, which suggests less cooperative thin filament activation and reduced recruitment of non-cycling cross-bridges into the pool of cycling cross-bridges, a process that would tend to depress both myocyte force and power. Regarding MyBP-C, PKA treatment of slow-twitch skeletal muscle fibers caused phosphorylation of MyBP-C (but not slow skeletal TnI (ssTnI)) and yielded faster loaded shortening velocity and ∼30% increase in power output. These results add novel insight into the molecular specificity by which the ß-adrenergic system regulates myofibrillar contractility and how attenuation of PKA-induced phosphorylation of cMyBP-C and cTnI may contribute to ventricular pump failure.