The work “Disruption of transverse‐tubular network reduces energy efficiency in cardiac muscle contraction” by Mellor et al¹ that appears in this issue of Acta Physiologica brings together the scientific interests and technical skills of two worlds: that of fine intact cardiac muscle energetics and mechanics (eg Tran et al² and other studies by Loiselle and his collaborators) and the one of disease‐associated cardiac t‐tubule remodelling (eg Crossman et al³ and other studies by Soeller and his collaborators). We would like to emphasize that the experimental data presented in Mellor et al¹ comes from the only laboratory in the world that, to date, is able to couple sophisticated force recordings to microcalorimetric measurements employing intact cardiac preparations of small size (ie single trabeculae) that are ideal in terms of fibre alignment, superfusate flow, etc for both mechanical and energetic assessments.² With their long‐standing experience, the authors show here how mechanical parameters (muscle stress, muscle shortening velocity and muscle power) and energetic parameters (activation heat, peak cross‐bridge heat and peak shortening heat) are altered following an acute disruption of t‐tubules. To acutely detached t‐tubules from the surface sarcolemma, the authors apply an osmotic shock protocol, developed in the 2000s on single cardiomyocytes,⁴ and more recently adapted for use on multicellular cardiac preparations.⁵ Formamide is an easily diffusible small molecule that, if added to the extracellular medium in high dosage, accumulates inside the cells without damaging them. When Formamide is removed from the extracellular environment (ie when the extracellular solution returns isoosmolar with the intracellular medium) the cells undergo a transient and rapid increase in volume (+17% approximately) which causes subsarcolemmal fragmentation of t‐tubules and their seal off from the surface. This technique has been shown to be effective in isolated cardiomyocytes but was thought to be unsuitable in multicellular preparations where the spatial packing of the myocytes themselves and the diffusional delays tend to prevent rapid volume changes. Ferrantini et al⁵ successfully adapted the formamide protocol to thin multicellular preparations (mainly right ventricular trabeculae with diameter <200 µm) and the procedure was subsequently adopted with no modifications and comparable results by Power et al⁶ In the present work, Mellor and collaborators¹ made the osmotic shock slightly more aggressive (2 mol/L Formamide applied for 20 minutes, instead of 1.5 mol/L applied for 15 minutes) and managed to effectively detubulate larger trabeculae dissected from the left ventricle. A slightly lower efficacy of the detubulation procedure (34% vs 58% decrease in mean TT power, the index used to quantify t‐tubule disruption) can be attributed to the larger preparation size and small differences in the protocol (eg the rate of superfusate flow for formamide removal, that is critical for t‐tubule detachment, was higher in Ferrantini et al⁵ (>5 vs 2 µL/s). What has been shown in all three studies by Mellor et al,¹ Ferrantini et al⁵ and Power et al⁶ using either confocal or two‐photon microscopy—with the latter being more appropriate for the higher z‐penetration depth—is that Formamide‐shock leads to a very inhomogeneous detachment of t‐tubules, resulting in a great variability in the degree of detubulation between different cells of the same trabecula, but also between different areas within the same cardiomyocyte. Far from being a limitation of the protocol, the use of formamide on multicellular preparations produces a t‐tubule distribution pattern that very closely mimics that found in cardiac pathological remodelling. In heart failure and other cardiac diseases (eg HCM), in fact, the loss of t‐tubules is patchy and non‐homogeneous.

The formamide‐induced acute detubulation model, unlike the disease‐associated t‐tubule remodelling, is an ideal platform to study the mechanical and energetic impact of t‐tubule network disruption, as the t‐tubule loss is not associated with any other type of damage. Detached tubules remain trapped inside, disconnected from the surface sarcolemma therefore not able to propagate the action potential (as confirmed with Voltage Sensitive Dyes,⁵), without leading to any structural alterations of the sarcoplasmic reticulum (SR) or surface sarcolemma. Can the acutely detached tubules reattach themselves to the surface? This possibility has been suggested by both Mellor et al¹ and Ferrantini et al⁵ because some mechanical parameters that initially undergo a variation, for example, the twitch duration, tend to return to baseline values about 20‐30 minutes after the osmotic shock. This may be related to the mechanical activity of the twitching muscle that favours t‐tubule resealing, but the possible recovery of t‐tubule connections to the surface was not adequately investigated by taking images at different times (10, 20 or >20 minutes) after the osmotic shock.

Following the detachment of t‐tubules, active stress is reduced, more pronouncedly at high pacing rates, and contraction kinetics are prolonged. These mechanical changes are robust findings because of the intrinsic experimental modality (coupled comparison between data obtained from the same muscle before and after detubulation) and are further strengthened by qualitatively and quantitatively concordant results in Mellor et al,¹ Power et al⁶ and Ferrantini et al.⁵

Mellor and collaborators¹ are the first to perform mechanical measurements on detubulated trabeculae during work‐loop contractions. In addition to reduced stress at any afterload, they show that following detubulation, trabeculae shorten less, at a reduced velocity and with reduced power. The reduced shortening at low afterloads, coupled with the reduced stress, results in reduced mechanical work output. Mellor and collaborators¹ also report that detubulation reduces activation heat by 20% (consistent with 21% reduction in the Ca²⁺ transient amplitude reported by Ferrantini et al⁵) and reduced cross‐bridge heat by 34% (in line with the approximately 30% reduction in active stress reported by Mellor et al,¹ Power et al,⁶ and Ferrantini et al⁵).

As the authors nicely explain, cross‐bridge heat can be partitioned into an isometric component and a shortening component, the proportions of which depend on afterload. Under isometric conditions, cross‐bridge heat consists entirely of isometric heat, and is quantified as the peak cross‐bridge heat, that is indeed reduced after t‐tubule detachment. Peak shortening heat at virtually zero afterload, instead, is not significantly changed, in line with no significant changes of peak shortening. In the detubulated myocardium, given an essentially unchanged maximal shortening velocity and a relative force‐velocity relationship that looks like the same as before acute detubulation, the reduction in power is much likely due to a reduction in the number of motors (while the properties of the motors are maintained the same); this reduction in the number of the motors is consistent with a reduction in the calcium activation following detubulation.

In fact, in simple terms, Mellor et al¹ show that changes occurring in the detubulated myocardium create a discrepancy between the marked decrease in force production by the myofilaments and the pretty modest decrease in their energy consumption. The authors did not investigate the mechanisms underlying this discrepancy that increases the energy cost of tension generation. One possible explanation is that the loss of tubules introduces a great inhomogeneity during calcium activation. Areas without tubules connected to the surface are activated with a delay, through a cascade‐like mechanism based on calcium diffusion/propagation from neighbouring areas (see for instance Ferrantini et al⁷ or other reviews on this topic). This propagated E‐C‐coupling mode, associated with t‐tubule disruption, causes the coexistence at the same time of in‐series sarcomeres with different levels of calcium activation (higher levels of activation where the tubule is maintained, lower levels where the tubule is disconnected), thus establishing a mechanism of inter‐sarcomere dynamics and inhomogeneity of sarcomere lengths during contraction. Of note, as Mellor et al¹ and Ferrantini et al⁵ observed, resting sarcomere length is not made inhomogeneous by the detubulation process per se (eg in Ferrantini et al⁵ sarcomere length fluctuations and its range of variability slightly increased during Formamide exposure but were normalized upon Formamide removal). During contraction, instead, the inhomogeneity of calcium activation levels and consequently sarcomere lengths—that particularly occurs during the twitch rising phase—leads to a reduction in the developed tension, while the consumption of ATP at any given tension level may increase (because of the non‐isometric sarcomere working conditions), creating the energetic discrepancies described by Mellor et al¹ This mechanism could be tested experimentally by making calcium activation within the detubulated preparations more homogeneous through positive inotropic interventions (eg β‐adrenergic stimulation, high extracellular calcium levels) or through the administration of pharmacological agents that resynchronize the calcium transient by favouring calcium propagation in the regions in which the tubules are disconnected (eg low doses of caffeine as in Ferrantini et al⁵). It would be of interest to see whether, under these conditions of increased/resynchronized calcium activation, the detubulated myocardium still shows a reduction in energy efficiency of about 30%, or if the energy efficiency is restored to values ​​that are closer to those of the healthy myocardium.

We believe that the work of Mellor et al¹ that exploits the detubulated myocardium as an experimental tool, is the first to demonstrate that t‐tubular disruption represents one structural pathophysiological pathway that can contribute to reduction in cardiac mechanical efficiency in disease conditions. The energy impairment introduced by t‐tubule disruption can be relevant in the pathophysiology of many cardiac conditions associated with t‐tubular structural remodelling and consequent loss of synchrony in calcium activation (eg Ferrantini et al⁷). First is Heart Failure, with an increased energy cost of contraction that notoriously derives from reduced myocardial force due to structural alterations (eg reduction of myofilaments density, increased presence of collagen) besides the increase in energy consumption required for calcium cycling and, likely, at myofilament level. Of high relevance are also Hypertrophic Cardiomyopathy, with cardiomyocyte energy depletion often due to altered cross‐bridge kinetics (because of the presence of a mutant sarcomeric protein or its lack of expression) (eg Piroddi et al⁸) and Dilated Cardiomyopathy, with reduced mechanical efficiency due to impaired tension transduction to the sarcolemma and the extracellular matrix. In all these conditions, a loss of t‐tubules and a desynchronization of the calcium transient have been reported and the pathophysiological mechanism described by Mellor et al,¹ that is, a “t‐tubule depletion”‐associated increase in the energy cost of tension generation, can represent an additional source of energy mismatch on top of others.

在本期《生理学报》上发表的Mellor等[ ^1]的“横管网络的破坏降低了心肌收缩的能量效率”^一书汇集了两个领域的科学兴趣和技术技能：完好无损的心肌能量学和力学（例如Tran等人²和Loiselle及其合作者的其他研究）以及与疾病相关的心脏t管重塑之一（例如Crossman等人³和Soeller及其合作者的其他研究）。我们想强调的是，Mellor等人^1中提供的实验数据来自世界上唯一的实验室，迄今为止，该实验室能够使用完整的小尺寸心脏制剂（即单小梁）将精密的力记录与微量热测量相结合，这在纤维排列，超融合流等方面都很理想机械和精力充沛的评估。²通过他们长期的经验，作者在这里展示了在剧烈破坏后，力学参数（肌肉应力，肌肉缩短速度和肌肉力量）和能量参数（激活热，峰值跨桥热和峰值缩短热）如何改变。 T型小管。对于从表面肉瘤到急性分离的T管，作者应用了2000年代开发的针对单个心肌细胞的渗透压休克方案，⁴最近更适合用于多细胞心脏制剂。⁵甲酰胺是一种易于扩散的小分子，如果以高剂量添加到细胞外培养基中，则会在细胞内积聚而不会损坏它们。当甲酰胺从细胞外环境中移出时（即当细胞外溶液与细胞内介质等渗时），细胞的体积会发生短暂而快速的增加（大约+ 17％），这会导致T形管的肌膜下碎裂，并使其密封。表面。已经表明该技术在分离的心肌细胞中是有效的，但是被认为不适用于多细胞制剂，在该多细胞制剂中，肌细胞本身的空间堆积和扩散延迟倾向于阻止体积的快速变化。Ferrantini等⁵成功地使甲酰胺方案适用于薄的多细胞制剂（主要是直径<200 µm的右心小梁），随后该程序被采用，未经修改，Power等人进行了比较⁶结果在目前的工作中，Mellor和合作者¹使渗透压休克更具侵略性（使用2 mol / L的甲酰胺20分钟，而不是使用1.5 mol / L的15分钟），并设法有效地使从左心室切开的较大小梁脱管。拔管手术的疗效略低（平均TT功率降低34％比58％，用于量化T管破裂的指标）可以归因于较大的制剂尺寸和方案中的细微差异（例如，超融合液的速率） Ferrantini等人[ ⁵ ]对T管分离至关重要的去除甲酰胺的流量较高（> 5 vs 2 µL / s），Mellor等人[ ^1]， Ferrantini等人[ ^5]和电源等⁶使用共聚焦或双光子显微镜（后者更适合较高的z穿透深度）是因为甲酰胺冲击会导致t形小管非常不均匀地脱离，从而导致两个管之间的脱管程度差异很大同一小梁的不同细胞，以及同一心肌细胞内不同区域之间的细胞。甲酰胺在多细胞制剂上的使用绝非协议的限制，它产生的肾小管分布模式非常类似于心脏病理重塑中的模式。实际上，在心力衰竭和其他心脏病（例如HCM）中，t管的丢失是零散的和不均匀的。

与疾病相关的t管重塑不同，甲酰胺诱导的急性脱管模型是研究t管网络破坏的机械和能量影响的理想平台，因为t管丢失与任何其他类型的损伤。分离的小管仍留在内部，与表面肌膜分离，因此不能传播动作电位（如电压敏感染料⁵所证实），而不会导致肌浆网（SR）或表面肌膜的任何结构改变。急性分离的小管是否可以重新附着在表面上？Mellor等人¹和Ferrantini等人⁵都提出了这种可能性。因为一些最初发生变化的机械参数（例如抽搐持续时间）在渗透性休克后约20-30分钟趋于恢复到基线值。这可能与抽搐肌肉的机械活动有关，这种机械运动有利于T管重新密封，但是通过在不同时间（10、20或> 20分钟）拍摄图像，未能充分研究到T管连接至表面的可能恢复。渗透性休克后。

T形管分离后，活动应力降低，尤其是在高起搏速率下，显着降低，并且收缩动力学得到延长。这些机械变化是可靠的发现，因为它们具有内在的实验方式（在拔管之前和之后从同一块肌肉获得的数据之间进行了比较比较），并且在定性和定量上一致的结果进一步得到了巩固（Mellor等人¹ Power等人⁶和Ferrantini等人）。等 ⁵

Mellor和合作者¹率先在工作循环收缩期间对脱管小梁进行机械测量。除了在任何后负荷下降低应力外，它们还表明，在拔管后，小梁缩短的时间更少，速度降低，功率降低。低后负荷时缩短的缩短时间减少，应力减小，导致机械功输出减少。Mellor和合作者¹还报告说，拔管可将激活热降低20％（与Ferrantini等人⁵报告的Ca ²⁺瞬态振幅降低21％相一致），并将跨桥热降低34％（与大约30一致）。 Mellor等[ ^1]报告的活动压力减少百分比Power等，⁶和Ferrantini等，⁵）。

正如作者很好地解释的那样，跨桥热可分为等距分量和缩短分量，其比例取决于后负荷。在等距条件下，跨桥热量完全由等距热量组成，并量化为峰值跨桥热量，实际上在T型管分离后会降低。相反，在几乎为零的后负荷下，峰缩短热没有明显改变，这与峰缩短没有明显变化相一致。在拔管的心肌中，假定基本不变的最大缩短速度和相对力-速度关系看起来与急性拔管之前相同，则功率降低很可能是由于电动机数量的减少而导致的。电机保持相同）；

实际上，简而言之，Mellor等人[ ^1]表明，在拔管的心肌中发生的变化在肌丝产生的力显着下降与能量消耗的适度下降之间产生了差异。作者没有研究这种差异潜在的机制，这种机制会增加产生张力的能源成本。一种可能的解释是，肾小管的丢失在钙激活过程中引入了很大的不均匀性。通过基于钙从邻近区域扩散/传播的级联机制，延迟激活了没有小管连接到表面的区域（例如，参见Ferrantini等人⁷或其他有关此主题的评论）。这传播了E-C偶联模式与T管破裂相关，导致同时发生具有不同钙激活水平的系列肉瘤并存（高水平的激活可维持小管，较低的水平可断开小管） ），从而建立了肌间动力学和收缩过程中肌节长度不均匀的机制。值得注意的是，如Mellor等人¹和Ferrantini等人^5所观察到的那样，静息肌节长度不会因拔管过程本身而变得不均匀（例如，在Ferrantini等人^5中）甲酰胺暴露期间肌节长度波动及其变异范围略有增加，但在甲酰胺去除后已恢复正常。相反，在收缩过程中，钙激活水平的不均匀性以及随之而来的肌小节长度（尤其是在抽搐上升阶段发生）会导致所产生的张力降低，而在任何给定的张力水平下，ATP的消耗都可能会增加（由于非等距肌节的工作条件），产生了Mellor等人¹描述的高能差异可以通过正性肌力干预（例如β-肾上腺素刺激，高细胞外钙水平）或通过使用通过促进钙在钙中的钙传播来使钙瞬变重新同步的药理剂，使脱管制剂中的钙活化更加均一，以实验方式测试这种机制。小管断开的区域（如Ferrantini等[ ⁵ ]中的低剂量咖啡因）。有趣的是，在这些钙激活增加/重新同步的条件下，拔管的心肌是否仍显示出约30％的能量效率降低，或者能量效率是否恢复到更接近那些值的值。健康的心肌。

我们认为，Mellor等人¹的研究以去管的心肌为实验工具，是第一个证明T管破裂代表一种结构病理生理途径的研究，该途径可有助于降低疾病状态下的心脏机械效率。T管破坏引起的能量损伤可能与许多与T管结构重塑以及随之而来的钙激活丧失同步性相关的心脏疾病的病理生理学有关（例如Ferrantini等人⁷）。首先是心力衰竭，除结构循环（例如钙循环）所需的能量消耗增加外，收缩的能量成本增加是由于结构改变（例如，肌丝密度降低，胶原蛋白的存在增加）而导致的心肌力降低而引起的。肌丝水平。肥厚型心肌病也具有高度相关性，心肌细胞能量耗竭通常归因于跨桥动力学的改变（由于突变型肌节蛋白的存在或其缺乏表达）（例如，Piroddi等人^8））和扩张型心肌病，由于向肌膜和细胞外基质的张力传导受损，机械效率降低。在所有这些情况下，已经报告了t小管丢失和钙瞬变失同步，Mellor等人[ ^1]描述了其病理生理机制，即“ t小管耗竭”导致能量消耗增加。张力的产生，可能是其他原因导致能量失配的另一个原因。