Regulation of cardiomyocyte t-tubule structure by preload and afterload: Roles in cardiac compensation and decompensation.

Mechanical load is a potent regulator of cardiac structure and function. Although high workload during heart failure is associated with disruption of cardiomyocyte t-tubules and Ca2+ homeostasis, it remains unclear whether changes in preload and afterload may promote adaptive t-tubule remodelling. We examined this issue by first investigating isolated effects of stepwise increases in load in cultured rat papillary muscles. Both preload and afterload increases produced a biphasic response, with the highest t-tubule densities observed at moderate loads, whereas excessively low and high loads resulted in low t-tubule levels. To determine the baseline position of the heart on this bell-shaped curve, mice were subjected to mildly elevated preload or afterload (1 week of aortic shunt or banding). Both interventions resulted in compensated cardiac function linked to increased t-tubule density, consistent with ascension up the rising limb of the curve. Similar t-tubule proliferation was observed in human patients with moderately increased preload or afterload (mitral valve regurgitation, aortic stenosis). T-tubule growth was associated with larger Ca2+ transients, linked to upregulation of L-type Ca2+ channels, Na+-Ca2+ exchanger, mechanosensors and regulators of t-tubule structure. By contrast, marked elevation of cardiac load in rodents and patients advanced the heart down the declining limb of the t-tubule-load relationship. This bell-shaped relationship was lost in the absence of electrical stimulation, indicating a key role of systolic stress in controlling t-tubule plasticity. In conclusion, modest augmentation of workload promotes compensatory increases in t-tubule density and Ca2+ cycling, whereas this adaptation is reversed in overloaded hearts during heart failure progression. KEY POINTS: Excised papillary muscle experiments demonstrated a bell-shaped relationship between cardiomyocyte t-tubule density and workload (preload or afterload), which was only present when muscles were electrically stimulated. The in vivo heart at baseline is positioned on the rising phase of this curve because moderate increases in preload (mice with brief aortic shunt surgery, patients with mitral valve regurgitation) resulted in t-tubule growth. Moderate increases in afterload (mice and patients with mild aortic banding/stenosis) similarly increased t-tubule density. T-tubule proliferation was associated with larger Ca2+ transients, with upregulation of the L-type Ca2+ channel, Na+-Ca2+ exchanger, mechanosensors and regulators of t-tubule structure. By contrast, marked elevation of cardiac load in rodents and patients placed the heart on the declining phase of the t-tubule-load relationship, promoting heart failure progression. The dependence of t-tubule structure on preload and afterload thus enables both compensatory and maladaptive remodelling, in rodents and humans.

BIN1, Myotubularin, and Dynamin-2 Coordinate T-Tubule Growth in Cardiomyocytes.

BACKGROUND: Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca2+ homeostasis. The membrane bending and scaffolding protein BIN1 (bridging integrator 1) has been implicated in this process. However, it is unclear which of the various reported BIN1 isoforms are involved, and whether BIN1 function is regulated by its putative binding partners MTM1 (myotubularin), a phosphoinositide 3'-phosphatase, and DNM2 (dynamin-2), a GTPase believed to mediate membrane fission. METHODS: We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca2+ release was recorded using Fluo-4. RESULTS: We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca2+ release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms. CONCLUSIONS: These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes.

Hypokalemia Promotes Arrhythmia by Distinct Mechanisms in Atrial and Ventricular Myocytes.

RATIONALE: Hypokalemia occurs in up to 20% of hospitalized patients and is associated with increased incidence of ventricular and atrial fibrillation. It is unclear whether these differing types of arrhythmia result from direct and perhaps distinct effects of hypokalemia on cardiomyocytes. OBJECTIVE: To investigate proarrhythmic mechanisms of hypokalemia in ventricular and atrial myocytes. METHODS AND RESULTS: Experiments were performed in isolated rat myocytes exposed to simulated hypokalemia conditions (reduction of extracellular [K+] from 5.0 to 2.7 mmol/L) and supported by mathematical modeling studies. Ventricular cells subjected to hypokalemia exhibited Ca2+ overload and increased generation of both spontaneous Ca2+ waves and delayed afterdepolarizations. However, similar Ca2+-dependent spontaneous activity during hypokalemia was only observed in a minority of atrial cells that were observed to contain t-tubules. This effect was attributed to close functional pairing of the Na+-K+ ATPase and Na+-Ca2+ exchanger proteins within these structures, as reduction in Na+ pump activity locally inhibited Ca2+ extrusion. Ventricular myocytes and tubulated atrial myocytes additionally exhibited early afterdepolarizations during hypokalemia, associated with Ca2+ overload. However, early afterdepolarizations also occurred in untubulated atrial cells, despite Ca2+ quiescence. These phase-3 early afterdepolarizations were rather linked to reactivation of nonequilibrium Na+ current, as they were rapidly blocked by tetrodotoxin. Na+ current-driven early afterdepolarizations in untubulated atrial cells were enabled by membrane hyperpolarization during hypokalemia and short action potential configurations. Brief action potentials were in turn maintained by ultra-rapid K+ current (IKur); a current which was found to be absent in tubulated atrial myocytes and ventricular myocytes. CONCLUSIONS: Distinct mechanisms underlie hypokalemia-induced arrhythmia in the ventricle and atrium but also vary between atrial myocytes depending on subcellular structure and electrophysiology.

A Matched-Filter-Based Algorithm for Subcellular Classification of T-System in Cardiac Tissues.

In mammalian ventricular cardiomyocytes, invaginations of the surface membrane form the transverse tubular system (T-system), which consists of transverse tubules (TTs) that align with sarcomeres and Z-lines as well as longitudinal tubules (LTs) that are present between Z-lines in some species. In many cardiac disease etiologies, the T-system is perturbed, which is believed to promote spatially heterogeneous, dyssynchronous Ca2+ release and inefficient contraction. In general, T-system characterization approaches have been directed primarily at isolated cells and do not detect subcellular T-system heterogeneity. Here, we present MatchedMyo, a matched-filter-based algorithm for subcellular T-system characterization in isolated cardiomyocytes and millimeter-scale myocardial sections. The algorithm utilizes "filters" representative of TTs, LTs, and T-system absence. Application of the algorithm to cardiomyocytes isolated from rat disease models of myocardial infarction (MI), dilated cardiomyopathy induced via aortic banding, and sham surgery confirmed and quantified heterogeneous T-system structure and remodeling. Cardiomyocytes from post-MI hearts exhibited increasing T-system disarray as proximity to the infarct increased. We found significant (p < 0.05, Welch's t-test) increases in LT density within cardiomyocytes proximal to the infarct (12 ± 3%, data reported as mean ± SD, n = 3) versus sham (4 ± 2%, n = 5), but not distal to the infarct (7 ± 1%, n = 3). The algorithm also detected decreases in TTs within 5° of the myocyte minor axis for isolated aortic banding (36 ± 9%, n = 3) and MI cardiomyocytes located intermediate (37 ± 4%, n = 3) and proximal (34 ± 4%, n = 3) to the infarct versus sham (57 ± 12%, n = 5). Application of bootstrapping to rabbit MI tissue revealed distal sections comprised 18.9 ± 1.0% TTs, whereas proximal sections comprised 10.1 ± 0.8% TTs (p < 0.05), a 46.6% decrease. The matched-filter approach therefore provides a robust and scalable technique for T-system characterization from isolated cells through millimeter-scale myocardial sections.

Regulation of Cardiomyocyte T-Tubular Structure: Opportunities for Therapy.

PURPOSE OF REVIEW: Membrane invaginations called t-tubules play an integral role in triggering cardiomyocyte contraction, and their disruption during diseases such as heart failure critically impairs cardiac performance. In this review, we outline the growing understanding of the malleability of t-tubule structure and function, and highlight emerging t-tubule regulators which may be exploited for novel therapies. RECENT FINDINGS: New technologies are revealing the nanometer scale organization of t-tubules, and their functional junctions with the sarcoplasmic reticulum called dyads, which generate Ca2+ sparks. Recent data have indicated that the dyadic anchoring protein junctophilin-2, and the membrane-bending protein BIN1 are key regulators of dyadic formation and maintenance. While the underlying signals which control expression and localization of these proteins remain unclear, accumulating data support an important role of myocardial workload. Although t-tubule alterations are believed to be a key cause of heart failure, the plasticity of these structures also creates an opportunity for therapy. Promising recent data suggest that such therapies may specifically target junctophilin-2, BIN1, and/or mechanotransduction.