Nonclinical evaluation of chronic cardiac contractility modulation on 3D human engineered cardiac tissues.

INTRODUCTION: Cardiac contractility modulation (CCM) is a medical device-based therapy delivering non-excitatory electrical stimulations to the heart to enhance cardiac function in heart failure (HF) patients. The lack of human in vitro tools to assess CCM hinders our understanding of CCM mechanisms of action. Here, we introduce a novel chronic (i.e., 2-day) in vitro CCM assay to evaluate the effects of CCM in a human 3D microphysiological system consisting of engineered cardiac tissues (ECTs). METHODS: Cryopreserved human induced pluripotent stem cell-derived cardiomyocytes were used to generate 3D ECTs. The ECTs were cultured, incorporating human primary ventricular cardiac fibroblasts and a fibrin-based gel. Electrical stimulation was applied using two separate pulse generators for the CCM group and control group. Contractile properties and intracellular calcium were measured, and a cardiac gene quantitative PCR screen was conducted. RESULTS: Chronic CCM increased contraction amplitude and duration, enhanced intracellular calcium transient amplitude, and altered gene expression related to HF (i.e., natriuretic peptide B, NPPB) and excitation-contraction coupling (i.e., sodium-calcium exchanger, SLC8). CONCLUSION: These data represent the first study of chronic CCM in a 3D ECT model, providing a nonclinical tool to assess the effects of cardiac electrophysiology medical device signals complementing in vivo animal studies. The methodology established a standardized 3D ECT-based in vitro testbed for chronic CCM, allowing evaluation of physiological and molecular effects on human cardiac tissues.

Lycium barbarum polysaccharides restore adverse structural remodelling and cardiac contractile dysfunction induced by overexpression of microRNA-1.

MicroRNA-1 (miR-1) stands out as the most prominent microRNA (miRNA) in regulating cardiac function and has been perceived as a new potential therapeutic target. Lycium barbarum polysaccharides (LBPs) are major active constituents of the traditional Chinese medicine based on L. barbarum. The purpose of this study was to exploit the cardioprotective effect and molecular mechanism of LBPs underlying heart failure. We found that LBPs significantly reduced the expression of myocardial miR-1. LBPs improved the abnormal ECG and indexes of cardiac functions in P-V loop detection in transgenic (Tg) mice with miR-1 overexpression. LBPs recovered morphological changes in sarcomeric assembly, intercalated disc and gap junction. LBPs reversed the reductions of CaM and cMLCK, the proteins targeted by miR-1. Similar trends were also obtained in their downstream effectors including the phosphorylation of MLC2v and both total level and phosphorylation of CaMKII and cMyBP-C. Collectively, LBPs restored adverse structural remodelling and improved cardiac contractile dysfunction induced by overexpression of miR-1. One of the plausible mechanisms was that LBPs down-regulated miR-1 expression and consequently reversed miR-1-induced repression of target proteins relevant to myocardial contractibility. LBPs could serve as a new, at least a very useful adjunctive, candidate for prevention and therapy of heart failure.

Multifocal ectopic Purkinje-related premature contractions: a new SCN5A-related cardiac channelopathy.

OBJECTIVES: The aim of this study was to describe a new familial cardiac phenotype and to elucidate the electrophysiological mechanism responsible for the disease. BACKGROUND: Mutations in several genes encoding ion channels, especially SCN5A, have emerged as the basis for a variety of inherited cardiac arrhythmias. METHODS: Three unrelated families comprising 21 individuals affected by multifocal ectopic Purkinje-related premature contractions (MEPPC) characterized by narrow junctional and rare sinus beats competing with numerous premature ventricular contractions with right and/or left bundle branch block patterns were identified. RESULTS: Dilated cardiomyopathy was identified in 6 patients, atrial arrhythmias were detected in 9 patients, and sudden death was reported in 5 individuals. Invasive electrophysiological studies demonstrated that premature ventricular complexes originated from the Purkinje tissue. Hydroquinidine treatment dramatically decreased the number of premature ventricular complexes. It normalized the contractile function in 2 patients. All the affected subjects carried the c.665G>A transition in the SCN5A gene. Patch-clamp studies of resulting p.Arg222Gln (R222Q) Nav1.5 revealed a net gain of function of the sodium channel, leading, in silico, to incomplete repolarization in Purkinje cells responsible for premature ventricular action potentials. In vitro and in silico studies recapitulated the normalization of the ventricular action potentials in the presence of quinidine. CONCLUSIONS: A new SCN5A-related cardiac syndrome, MEPPC, was identified. The SCN5A mutation leads to a gain of function of the sodium channel responsible for hyperexcitability of the fascicular-Purkinje system. The MEPPC syndrome is responsive to hydroquinidine.

Short-chain fatty acid propionate alleviates Akt2 knockout-induced myocardial contractile dysfunction.

BACKGROUND AND AIMS. Dysregulation of Akt has been implicated in diseases such as cancer and diabetes, although little is known about the role of Akt deficiency on cardiomyocyte contractile function. This study was designed to examine the effect of Akt2 knockout-induced cardiomyocyte contractile response and the effect of dietary supplementation of short-chain fatty acid propionate on Akt2 knockout-induced cardiac dysfunction, if any. METHODS AND RESULTS. Adult male wild-type (WT) and Akt2 knockout mice were treated with propionate (0.3 g/kg, p.o.) or vehicle for 7 days. Oral glucose tolerance test (OGTT) was performed. Cardiomyocyte contractile function and mitochondrial membrane potential were assessed. Expression of insulin-signaling molecules Akt, PTEN, GSK3ß, and eNOS receptors for short-chain fatty acids GPR41, and GPR43 as well as protein phosphatase PP2AA, PP2AB, PP2C were evaluated using Western blot analysis. Our results revealed that Akt2 knockout led to overt glucose intolerance, compromised cardiomyocyte contractile function (reduced peak shortening and maximal velocity of shortening/relengthening as well as prolonged relengthening), loss of mitochondrial membrane potential, decreased GPR41 and elevated GPR43 expression, all of which, with the exception of glucose intolerance and elevated GPR43 level, were significantly attenuated by propionate. Neither Akt2 knockout nor propionate affected the expression of protein phosphatases, eNOS, pan, and phosphorylated PTEN and GSK3ß. CONCLUSIONS. Taken together, these data depicted that Akt2 knockout may elicit cardiomyocyte contractile and mitochondrial defects and a beneficial role of propionate or short-chain fatty acids against Akt2 deficiency-induced cardiac anomalies.

BIN1 localizes the L-type calcium channel to cardiac T-tubules.

The BAR domain protein superfamily is involved in membrane invagination and endocytosis, but its role in organizing membrane proteins has not been explored. In particular, the membrane scaffolding protein BIN1 functions to initiate T-tubule genesis in skeletal muscle cells. Constitutive knockdown of BIN1 in mice is perinatal lethal, which is associated with an induced dilated hypertrophic cardiomyopathy. However, the functional role of BIN1 in cardiomyocytes is not known. An important function of cardiac T-tubules is to allow L-type calcium channels (Cav1.2) to be in close proximity to sarcoplasmic reticulum-based ryanodine receptors to initiate the intracellular calcium transient. Efficient excitation-contraction (EC) coupling and normal cardiac contractility depend upon Cav1.2 localization to T-tubules. We hypothesized that BIN1 not only exists at cardiac T-tubules, but it also localizes Cav1.2 to these membrane structures. We report that BIN1 localizes to cardiac T-tubules and clusters there with Cav1.2. Studies involve freshly acquired human and mouse adult cardiomyocytes using complementary immunocytochemistry, electron microscopy with dual immunogold labeling, and co-immunoprecipitation. Furthermore, we use surface biotinylation and live cell confocal and total internal fluorescence microscopy imaging in cardiomyocytes and cell lines to explore delivery of Cav1.2 to BIN1 structures. We find visually and quantitatively that dynamic microtubules are tethered to membrane scaffolded by BIN1, allowing targeted delivery of Cav1.2 from the microtubules to the associated membrane. Since Cav1.2 delivery to BIN1 occurs in reductionist non-myocyte cell lines, we find that other myocyte-specific structures are not essential and there is an intrinsic relationship between microtubule-based Cav1.2 delivery and its BIN1 scaffold. In differentiated mouse cardiomyocytes, knockdown of BIN1 reduces surface Cav1.2 and delays development of the calcium transient, indicating that Cav1.2 targeting to BIN1 is functionally important to cardiac calcium signaling. We have identified that membrane-associated BIN1 not only induces membrane curvature but can direct specific antegrade delivery of microtubule-transported membrane proteins. Furthermore, this paradigm provides a microtubule and BIN1-dependent mechanism of Cav1.2 delivery to T-tubules. This novel Cav1.2 trafficking pathway should serve as an important regulatory aspect of EC coupling, affecting cardiac contractility in mammalian hearts.

miR-1 overexpression enhances Ca(2+) release and promotes cardiac arrhythmogenesis by targeting PP2A regulatory subunit B56alpha and causing CaMKII-dependent hyperphosphorylation of RyR2.

MicroRNAs are small endogenous noncoding RNAs that regulate protein expression by hybridization to imprecise complementary sequences of target mRNAs. Changes in abundance of muscle-specific microRNA, miR-1, have been implicated in cardiac disease, including arrhythmia and heart failure. However, the specific molecular targets and cellular mechanisms involved in the action of miR-1 in the heart are only beginning to emerge. In this study we investigated the effects of increased expression of miR-1 on excitation-contraction coupling and Ca(2+) cycling in rat ventricular myocytes using methods of electrophysiology, Ca(2+) imaging and quantitative immunoblotting. Adenoviral-mediated overexpression of miR-1 in myocytes resulted in a marked increase in the amplitude of the inward Ca(2+) current, flattening of Ca(2+) transients voltage dependence, and enhanced frequency of spontaneous Ca(2+) sparks while reducing the sarcoplasmic reticulum Ca(2+) content as compared with control. In the presence of isoproterenol, rhythmically paced, miR-1-overexpressing myocytes exhibited spontaneous arrhythmogenic oscillations of intracellular Ca(2+), events that occurred rarely in control myocytes under the same conditions. The effects of miR-1 were completely reversed by the CaMKII inhibitor KN93. Although phosphorylation of phospholamban was not altered, miR-1 overexpression increased phosphorylation of the ryanodine receptor (RyR2) at S2814 (Ca(2+)/calmodulin-dependent protein kinase) but not at S2808 (protein kinase A). Overexpression of miR-1 was accompanied by a selective decrease in expression of the protein phosphatase PP2A regulatory subunit B56alpha involved in PP2A targeting to specialized subcellular domains. We conclude that miR-1 enhances cardiac excitation-contraction coupling by selectively increasing phosphorylation of the L-type and RyR2 channels via disrupting localization of PP2A activity to these channels.

Protein kinase A-mediated phosphorylation contributes to enhanced contraction observed in mice that overexpress beta-adrenergic receptor kinase-1.

Transgenic mice with cardiac specific overexpression of beta-adrenergic receptor kinase-1 (betaARK-1) exhibit reduced contractility in the presence of adrenergic stimulation. However, whether contractility is altered in the absence of exogenous agonist is not clear. Effects of betaARK-1 overexpression on contraction were examined in mouse ventricular myocytes, studied at 37 degrees C, in the absence of adrenergic stimulation. In myocytes voltage-clamped with microelectrodes (18-26 MOmega; 2.7 M KCl) to minimize intracellular dialysis, contractions were significantly larger in betaARK-1 cells than in wild-type myocytes. In contrast, when cells were dialyzed with patch pipette solution (1-3 MOmega; 0 mM NaCl, 70 mM KCl, 70 mM potassium aspartate, 4 mM MgATP, 1 mM MgCl(2), 2.5 mM KH(2)PO(4), 0.12 mM CaCl(2), 0.5 mM EGTA, and 10 mM HEPES), the extent of cell shortening was similar in wild-type and betaARK-1 myocytes. Furthermore, when cells were dialyzed with solutions that contained phosphodiesterase-sensitive sodium-cAMP (50 microM), the extent of cell shortening was similar in wild-type and betaARK-1 myocytes. However, when patch solutions were supplemented with phosphodiesterase-resistant 8-bromo-cAMP (50 muM), contractions were larger in betaARK-1 than wild-type cells. This difference was eliminated by the protein kinase A inhibitor N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89). Interestingly, Ca(2+) current amplitudes and inactivation rates were similar in betaARK-1 and wild-type cells in all experiments. These results suggest components of the adenylyl cyclase-protein kinase A pathway are sensitized by chronically increased betaARK-1 activity, which may augment contractile function in the absence of exogenous agonist. Thus, changes in contractile function in myocytes from failing hearts may reflect, in part, effects of chronic up-regulation of betaARK-1 on the cAMP-protein kinase A pathway.

Remodeling of excitation-contraction coupling in transgenic mice expressing ATP-insensitive sarcolemmal KATP channels.

Reducing the ATP sensitivity of the sarcolemmal ATP-sensitive K(+) (K(ATP)) channel is predicted to lead to active channels in normal metabolic conditions and hence cause shortened ventricular action potentials and reduced myocardial inotropy. We generated transgenic (TG) mice that express an ATP-insensitive K(ATP) channel mutant [Kir6.2(deltaN2-30,K185Q)] under transcriptional control of the alpha-myosin heavy chain promoter. Strikingly, myocyte contraction amplitude was increased in TG myocytes (15.68 +/- 1.15% vs. 10.96 +/- 1.49%), even though K(ATP) channels in TG myocytes are very insensitive to inhibitory ATP. Under normal metabolic conditions, steady-state outward K(+) currents measured under whole cell voltage clamp were elevated in TG myocytes, consistent with threshold K(ATP) activation, but neither the monophasic action potential measured in isolated hearts nor transmembrane action potential measured in right ventricular muscle preparations were shortened at physiological pacing cycles. Taken together, these results suggest that there is a compensatory remodeling of excitation-contraction coupling in TG myocytes. Whereas there were no obvious differences in other K(+) conductances, peak L-type Ca(2+) current (I(Ca)) density (-16.42 +/- 2.37 pA/pF) in the TG was increased compared with the wild type (-8.43 +/- 1.01 pA/pF). Isoproterenol approximately doubled both I(Ca) and contraction amplitude in wild-type myocytes but failed to induce a significant increase in TG myocytes. Baseline and isoproterenol-stimulated cAMP concentrations were not different in wild-type and TG hearts, suggesting that the enhancement of I(Ca) in the latter does not result from elevated cAMP. Collectively, the data demonstrate that a compensatory increase in I(Ca) counteracts a mild activation of ATP-insensitive K(ATP) channels to maintain the action potential duration and elevate the inotropic state of TG hearts.

Myosin light chain replacement in the heart.

Myosin-actin cross-bridge kinetics are an important determinant for cardiac systolic and diastolic function. We compared the effects of myosin light chain substitutions on the ability of the fibers to contract in response to calcium and in their ability to produce power. Transgenesis was used to effect essentially complete replacement of the target contractile protein isoform specifically in the heart. Atrial and ventricular fibers derived from the various transgenic (TG) lines were skinned, and the force-velocity relationships, unloaded shortening velocities, and Ca(2+)-stimulated Mg(2+)-ATPase activities were determined. Replacement with an ectopic isoform resulted in significant changes in cross-bridge cycling kinetics but without any overt effects on morbidity or mortality. To confirm that this result was not light chain specific, a modified alpha-myosin heavy chain isoform that resulted in significant changes in force development was also engineered. The animals appeared healthy and have normal lifespans, and the changes in force development did not result in significant remodeling or overt hypertrophy. We conclude that myosin light chains can control aspects of cross-bridge cycling and alter force development. The myosin heavy chain data also show that changes in the kinetics of force development and power output do not necessarily lead to activation of the hypertrophic response or significant cardiac remodeling.

Direct, convergent hypersensitivity of calcium-activated force generation produced by hypertrophic cardiomyopathy mutant alpha-tropomyosins in adult cardiac myocytes.

Familial hypertrophic cardiomyopathy is a clinically and genetically diverse autosomal dominant disorder characterized by ventricular hypertrophy and myocyte disarray in the absence of known hypertrophic stimuli. It has been linked to many cardiac contractile proteins, including four point mutations in alpha-tropomyosin (Tm). Here we use adenoviral-mediated gene transfer into adult cardiac myocytes in vitro to show that all four hypertrophic cardiomyopathy alpha-Tm proteins can be expressed and incorporated into normal sarcomeric structures in cardiac myocytes at similar levels as normal alpha-Tm proteins after 5-6 days in culture. Isometric force recordings of single cardiac myocytes demonstrated inappropriate increased force output at submaximal calcium concentration with a specific mutant allele hierarchy. These data indicate that the severity of direct calcium-sensitizing effect of mutations in alpha-Tm may predict the clinical severity of mutant alpha-Tm-associated hypertrophic cardiomyopathy.