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.

Advanced age and female sex protect cerebral arteries from mitochondrial depolarization and apoptosis during acute oxidative stress.

Aging increases reactive oxygen species (ROS) which can impair vascular function and contribute to brain injury. However, aging can also promote resilience to acute oxidative stress. Therefore, we tested the hypothesis that advanced age protects smooth muscle cells (SMCs) and endothelial cells (ECs) of posterior cerebral arteries (PCAs; diameter, ∼80 µm) during exposure to H2 O2 . PCAs from young (4-6 months) and old (20-26 months) male and female C57BL/6 mice were isolated and pressurized (~70 mm Hg) to evaluate cell death, mitochondrial membrane potential (ΔΨm ), ROS production, and [Ca2+ ]i in response to H2 O2 (200 µM, 50 min). SMC death and ΔΨm depolarization were greater in PCAs from males vs. females. Aging increased ROS in PCAs from both sexes but increased SMC resilience to death only in males. Inhibiting TRPV4 channels with HC-067047 (1 µM) or Src kinases with SU6656 (10 µM) reduced Ca2+ entry and SMC death to H2 O2 most effectively in PCAs from young males. Activating TRPV4 channels with GSK1016790A (50 nM) evoked greater Ca2+ influx in SMCs and ECs of PCAs from young vs. old mice but did not induce cell death. However, when combined with H2 O2 , TRPV4 activation exacerbated EC death. Activating Src kinases with spermidine (100 µM) increased Ca2+ influx in PCAs from males vs. females with minimal cell death. We conclude that in males, chronic oxidative stress during aging increases the resilience of cerebral arteries, which contrasts with inherent protection in females. Findings implicate TRP channels and Src kinases as targets to limit vascular damage to acute oxidative injury.

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.

Cardiac TRPV4 channels.

Transient Receptor Potential Vanilloid 4 (TRPV4) is expressed in numerous cell types within the heart, yet the expression levels, subcellular localization, and functional relevance of TRPV4 in cardiac myocytes is under-appreciated. Recent data indicate a critical role of TRPV4 in both atrial and ventricular myocyte biology, with expression levels and channel function increasing following pathological scenarios including ischemia, myocardial infarction, mechanical stress, and inflammation. Excessive activation of TRPV4 at the cellular level contributes to enhanced Ca2+ entry which predisposes the cardiac myocyte to pro-arrhythmic Ca2+ overload and electrophysiological abnormalities. At the organ level, excessive TRPV4 activity associates with cardiac hypercontractility, cardiac damage, ventricular arrhythmia, and atrial fibrillation. This manuscript chapter describes the emerging literature on TRPV4 in cardiac myocytes in physiology and disease.

Distribution of cardiomyocyte-selective adeno-associated virus serotype 9 vectors in swine following intracoronary and intravenous infusion.

Limited reports exist regarding adeno-associated virus (AAV) biodistribution in swine. This study assessed biodistribution following antegrade intracoronary and intravenous delivery of two self-complementary serotype 9 AAV (AAV9sc) biologics designed to target signaling in the cardiomyocyte considered important for the development of heart failure. Under the control of a cardiomyocyte-specific promoter, AAV9sc.shmAKAP and AAV9sc.RBD express a small hairpin RNA for the perinuclear scaffold protein muscle A-kinase anchoring protein ß (mAKAPß) and an anchoring disruptor peptide for p90 ribosomal S6 kinase type 3 (RSK3), respectively. Quantitative PCR was used to assess viral genome (vg) delivery and transcript expression in Ossabaw and Yorkshire swine tissues. Myocardial viral delivery was 2-5 × 105 vg/µg genomic DNA (gDNA) for both infusion techniques at a dose ∼1013 vg/kg body wt, demonstrating delivery of ∼1-3 viral particles per cardiac diploid genome. Myocardial RNA levels for each expressed transgene were generally proportional to dose and genomic delivery, and comparable with levels for moderately expressed endogenous genes. Despite significant AAV9sc delivery to other tissues, including the liver, neither biologic induced toxic effects as assessed using functional, structural, and circulating cardiac and systemic markers. These results indicate successful targeted delivery of cardiomyocyte-selective viral vectors in swine without negative side effects, an important step in establishing efficacy in a preclinical experimental setting.

Tempol Preserves Endothelial Progenitor Cells in Male Mice with Ambient Fine Particulate Matter Exposure.

Ambient fine particulate matter (PM) exposure associates with an increased risk of cardiovascular diseases (CVDs). Major sex differences between males and females exist in epidemiology, pathophysiology, and outcome of CVDs. Endothelial progenitor cells (EPCs) play a vital role in the development and progression of CVDs. PM exposure-induced reduction of EPCs is observed in male, not female, mice with increased reactive oxygen species (ROS) production and oxidative stress. The lung is considered an important source of ROS in mice with PM exposure. The aim of the present study was to investigate the sex differences in pulmonary superoxide dismutase (SOD) expression and ROS production, and to test the effect of SOD mimic Tempol on the populations of EPCs in mice with PM exposure. Both male and female C57BL/6 mice (8-10 weeks) were exposed to intranasal PM or vehicle for 6 weeks. Flow cytometry analysis demonstrated that PM exposure significantly decreased the levels of EPCs (CD34+/CD133+) in both blood and bone marrow with increased ROS production in males, but not in females. ELISA analysis showed higher levels of serum IL-6 and IL-1ßin males than in females. Pulmonary expression of the antioxidant enzyme SOD1 was significantly decreased in males after PM exposure, but not in females. Administration of the SOD mimic Tempol in male mice with PM exposure attenuated the production of ROS and inflammatory cytokines, and preserved EPC levels. These data indicated that PM exposure-induced reduction of EPC population in male mice may be due to decreased expression of pulmonary SOD1 in male mice.

Arrhythmogenesis in the aged heart following ischaemia-reperfusion: role of transient receptor potential vanilloid 4.

AIMS: Cardiomyocyte Ca2+ homoeostasis is altered with ageing and predisposes the heart to Ca2+ intolerance and arrhythmia. Transient receptor potential vanilloid 4 (TRPV4) is an osmotically activated cation channel with expression in cardiomyocytes of the aged heart. The objective of this study was to examine the role of TRPV4 in Ca2+ handling and arrhythmogenesis following ischaemia-reperfusion (I/R), a pathological scenario associated with osmotic stress. METHODS AND RESULTS: Cardiomyocyte membrane potential was monitored prior to and following I/R in Langendorff-perfused hearts of Aged (19-28 months) male and female C57BL/6 mice ± TRPV4 inhibition (1 µM HC067047, HC). Diastolic resting membrane potential was similar between Aged and Aged HC at baseline, but following I/R Aged exhibited depolarized diastolic membrane potential vs. Aged HC. The effects of TRPV4 on cardiomyocyte Ca2+ signalling following I/R were examined in isolated hearts of Aged cardiac-specific GCaMP6f mice (±HC) using high-speed confocal fluorescence microscopy, with cardiomyocytes of Aged exhibiting an increased incidence of pro-arrhythmic Ca2+ signalling vs. Aged HC. In the isolated cell environment, cardiomyocytes of Aged responded to sustained hypoosmotic stress (250mOsm) with an increase in Ca2+ transient amplitude (fluo-4) and higher incidence of pro-arrhythmic diastolic Ca2+ signals vs. Aged HC. Intracardiac electrocardiogram measurements in isolated hearts following I/R revealed an increased arrhythmia incidence, an accelerated time to ventricular arrhythmia, and increased arrhythmia score in Aged vs. Aged HC. Aged exhibited depolarized resting membrane potential, increased pro-arrhythmic diastolic Ca2+ signalling, and greater incidence of arrhythmia when compared with Young (3-5 months). CONCLUSION: TRPV4 contributes to pro-arrhythmic cardiomyocyte Ca2+ signalling, electrophysiological abnormalities, and ventricular arrhythmia in the aged mouse heart.

The right ventricular transcriptome signature in Ossabaw swine with cardiometabolic heart failure: implications for the coronary vasculature.

Heart failure (HF) patients with deteriorating right ventricular (RV) structure and function have a nearly twofold increased risk of death compared with those without. Despite the well-established clinical risk, few studies have examined the molecular signature associated with this HF condition. The purpose of this study was to integrate morphological, molecular, and functional data with the transcriptome data set in the RV of a preclinical model of cardiometabolic HF. Ossabaw swine were fed either normal diet without surgery (lean control, n = 5) or Western diet and aortic-banding (WD-AB; n = 4). Postmortem RV weight was increased and positively correlated with lung weight in the WD-AB group compared with CON. Total RNA-seq was performed and gene expression profiles were compared and analyzed using principal component analysis, weighted gene co-expression network analysis, module enrichment analysis, and ingenuity pathway analysis. Gene networks specifically associated with RV hypertrophic remodeling identified a hub gene in MAPK8 (or JNK1) that was associated with the selective induction of the extracellular matrix (ECM) component fibronectin. JNK1 and fibronectin protein were increased in the right coronary artery (RCA) of WD-AB animals and associated with a decrease in matrix metalloproteinase 14 protein, which specifically degrades fibronectin. RCA fibronectin content was correlated with increased vascular stiffness evident as a decreased elastin elastic modulus in WD-AB animals. In conclusion, this study establishes a molecular and transcriptome signature in the RV using Ossabaw swine with cardiometabolic HF. This signature was associated with altered ECM regulation and increased vascular stiffness in the RCA, with selective dysregulation of fibronectin.

Tissue-specific small heat shock protein 20 activation is not associated with traditional autophagy markers in Ossabaw swine with cardiometabolic heart failure.

The small heat shock protein 20 (HSPB6) emerges as a potential upstream mediator of autophagy. Although autophagy is linked to several clinical disorders, how HSPB6 and autophagy are regulated in the setting of heart failure (HF) remains unknown. The goal of this study was to assess the activation of the HSPB6 and its association with other well-established autophagy markers in central and peripheral tissues from a preclinical Ossabaw swine model of cardiometabolic HF induced by Western diet and chronic cardiac pressure overload. We hypothesized HSPB6 would be activated in central and peripheral tissues, stimulating autophagy. We found that autophagy in the heart is interrupted at various stages of the process in a chamber-specific manner. Protein levels of HSPB6, Beclin 1, and p62 are increased in the right ventricle, whereas only HSPB6 was increased in the left ventricle. Unlike the heart, samples from the triceps brachii long head showed only an increase in the protein level of p62, highlighting interesting central versus peripheral differences in autophagy regulation. In the right coronary artery, total HSPB6 protein expression was decreased and associated with an increase in LC3B-II/LC3B-I ratio, demonstrating a different mechanism of autophagy dysregulation in the coronary vasculature. Thus, contrary to our hypothesis, activation of HSPB6 was differentially regulated in a tissue-specific manner and observed in parallel with variable states of autophagy markers assessed by protein levels of LC3B, p62, and Beclin 1. Our data provide insight into how the HSPB6/autophagy axis is regulated in a preclinical swine model with potential relevance to heart failure with preserved ejection fraction.NEW & NOTEWORTHY Our study shows that the activation of HSPB6 is tissue specific and associated with variable states of downstream markers of autophagy in a unique preclinical swine model of cardiometabolic HF with potential relevance to HFpEF. These findings suggest that targeted approaches could be an important consideration regarding the development of drugs aimed at this intracellular recycling process.

Endothelial sodium channel activation promotes cardiac stiffness and diastolic dysfunction in Western diet fed female mice.

OBJECTIVE: Obesity is associated with myocardial fibrosis and impaired diastolic relaxation, abnormalities that are especially prevalent in women. Normal coronary vascular endothelial function is integral in mediating diastolic relaxation, and recent work suggests increased activation of the endothelial cell (EC) mineralocorticoid receptor (ECMR) is associated with impaired diastolic relaxation. As the endothelial Na+ channel (EnNaC) is a downstream target of the ECMR, we sought to determine whether EC-specific deletion of the critical alpha subunit, αEnNaC, would prevent diet induced-impairment of diastolic relaxation in female mice. METHODS AND MATERIALS: Female αEnNaC KO mice and littermate controls were fed a Western diet (WD) high in fat (46%), fructose corn syrup (17.5%) and sucrose (17.5%) for 12-16 weeks. Measurements were conducted for in vivo cardiac function, in vitro cardiomyocyte stiffness and EnNaC activity in primary cultured ECs. Additional biochemical studies examined indicators of oxidative stress, including aspects of antioxidant Nrf2 signaling, in cardiac tissue. RESULTS: Deletion of αEnNaC in female mice fed a WD significantly attenuated WD mediated impairment in diastolic relaxation. Improved cardiac relaxation was accompanied by decreased EnNaC-mediated Na+ currents in ECs and reduced myocardial oxidative stress. Further, deletion of αEnNaC prevented WD-mediated increases in isolated cardiomyocyte stiffness. CONCLUSION: Collectively, these findings support the notion that WD feeding in female mice promotes activation of EnNaC in the vasculature leading to increased cardiomyocyte stiffness and diastolic dysfunction.

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.

TRPV4 increases cardiomyocyte calcium cycling and contractility yet contributes to damage in the aged heart following hypoosmotic stress.

Aims: Cardiomyocyte Ca2+ homeostasis is altered with aging via poorly-understood mechanisms. The Transient Receptor Potential Vanilloid 4 (TRPV4) ion channel is an osmotically-activated Ca2+ channel, and there is limited information on the role of TRPV4 in cardiomyocytes. Our data show that TRPV4 protein expression increases in cardiomyocytes of the aged heart. The objective of this study was to examine the role of TRPV4 in cardiomyocyte Ca2+ homeostasis following hypoosmotic stress and to assess the contribution of TRPV4 to cardiac contractility and tissue damage following ischaemia-reperfusion (I/R), a pathological condition associated with cardiomyocyte osmotic stress. Methods and results: TRPV4 protein expression increased in cardiomyocytes of Aged (24-27 months) mice compared with Young (3-6 months) mice. Immunohistochemistry revealed TRPV4 localization to microtubules and the t-tubule network of cardiomyocytes of Aged mice, as well as in left ventricular myocardium of elderly patients undergoing surgical aortic valve replacement for aortic stenosis. Following hypoosmotic stress, cardiomyocytes of Aged, but not Young exhibited an increase in action-potential induced Ca2+ transients. This effect was mediated via increased sarcoplasmic reticulum Ca2+ content and facilitation of Ryanodine Receptor Ca2+ release and was prevented by TRPV4 antagonism (1 µmol/L HC067047). A similar hypoosmotic stress-induced facilitation of Ca2+ transients was observed in Young transgenic mice with inducible TRPV4 expression in cardiomyocytes. Following I/R, isolated hearts of Young mice with transgenic TRPV4 expression exhibited enhanced contractility vs. hearts of Young control mice. Similarly, hearts of Aged mice exhibited enhanced contractility vs. hearts of Aged TRPV4 knock-out (TRPV4-/-) mice. In Aged, pharmacological inhibition of TRPV4 (1 µmol/L, HC067047) prevented hypoosmotic stress-induced cardiomyocyte death and I/R-induced cardiac damage. Conclusions: Our findings provide a new mechanism for hypoosmotic stress-induced cardiomyocyte Ca2+ entry and cell damage in the aged heart. These finding have potential implications in treatment of elderly populations at increased risk of myocardial infarction and I/R injury.

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.

Cardiomyocyte Ca2+ homeostasis as a therapeutic target in heart failure with reduced and preserved ejection fraction.

Heart failure is a highly prevalent syndrome of multiple etiologies and associated comorbidities, and aberrant intracellular Ca2+ homeostasis is a hallmark finding in heart failure patients. The cyclical changes in Ca2+ concentration within cardiomyocytes control cycles of cardiac contraction and relaxation, and dysregulation of Ca2+ handling processes leads to systolic dysfunction, diastolic dysfunction, and adverse remodeling. For this reason, greater understanding of Ca2+ handling mechanisms in heart failure is critical for selection of appropriate treatment strategies. In this review, we summarize the mechanisms of altered Ca2+ handling in two subsets of heart failure, heart failure with reduced ejection fraction and heart failure with preserved ejection fraction, and outline current and experimental treatments that target cardiomyocyte Ca2+ handling processes.

Enhanced Development of Skeletal Myotubes from Porcine Induced Pluripotent Stem Cells.

The pig is recognized as a valuable model in biomedical research in addition to its agricultural importance. Here we describe a means for generating skeletal muscle efficiently from porcine induced pluripotent stem cells (piPSC) in vitro thereby providing a versatile platform for applications ranging from regenerative biology to the ex vivo cultivation of meat. The GSK3B inhibitor, CHIR99021 was employed to suppress apoptosis, elicit WNT signaling events and drive naïve-type piPSC along the mesoderm lineage, and, in combination with the DNA methylation inhibitor 5-aza-cytidine, to activate an early skeletal muscle transcription program. Terminal differentiation was then induced by activation of an ectopically expressed MYOD1. Myotubes, characterized by myofibril development and both spontaneous and stimuli-elicited excitation-contraction coupling cycles appeared within 11 days. Efficient lineage-specific differentiation was confirmed by uniform NCAM1 and myosin heavy chain expression. These results provide an approach for generating skeletal muscle that is potentially applicable to other pluripotent cell lines and to generating other forms of muscle.

Fascin2 regulates cisplatin-induced apoptosis in NRK-52E cells.

Previous studies have shown that the aging kidney has a marked loss of α(E)-catenin in proximal tubular epithelium. α-Catenin, a key regulator of the actin cytoskeleton, interacts with a variety of actin-binding proteins. Cisplatin-induced loss of fascin2, an actin bundling protein, was observed in cells with a stable knockdown of α(E)-catenin (C2 cells), as well as in aging (24 mon), but not young (4 mon), kidney. Fascin2 co-localized with α-catenin and the actin cytoskeleton in NRK-52E cells. Knockdown of fascin2 increased the susceptibility of tubular epithelial cells to cisplatin-induced injury. Overexpression of fascin2 in C2 cells restored actin stress fibers and attenuated the increased sensitivity of C2 cells to cisplatin-induced apoptosis. Interestingly, fascin2 overexpression attenuated cisplatin-induced mitochondrial dysfunction and oxidative stress in C2 cells. These data demonstrate that fascin2, a putative target of α(E)-catenin, may play important role in preventing cisplatin-induced acute kidney injury.

Prospect of gene therapy for cardiomyopathy in hereditary muscular dystrophy.

INTRODUCTION: Cardiac involvement is a common feature in muscular dystrophies. It presents as heart failure and/or arrhythmia. Traditionally, dystrophic cardiomyopathy is treated with symptom-relieving medications. Identification of disease-causing genes and investigation on pathogenic mechanisms have opened new opportunities to treat dystrophic cardiomyopathy with gene therapy. Replacing/repairing the mutated gene and/or targeting the pathogenic process/mechanisms using alternative genes may attenuate heart disease in muscular dystrophies. AREAS COVERED: Duchenne muscular dystrophy is the most common muscular dystrophy. Duchenne cardiomyopathy has been the primary focus of ongoing dystrophic cardiomyopathy gene therapy studies. Here, we use Duchenne cardiomyopathy gene therapy to showcase recent developments and to outline the path forward. We also discuss gene therapy status for cardiomyopathy associated with limb-girdle and congenital muscular dystrophies, and myotonic dystrophy. EXPERT OPINION: Gene therapy for dystrophic cardiomyopathy has taken a slow but steady path forward. Preclinical studies over the last decades have addressed many fundamental questions. Adeno-associated virus-mediated gene therapy has significantly improved the outcomes in rodent models of Duchenne and limb girdle muscular dystrophies. Validation of these encouraging results in large animal models will pave the way to future human trials.

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.