The field of cardiovascular research has benefitted from rapid developments in imaging technology over the last few decades. Accordingly, an ever growing number of large, multidimensional data sets have begun to appear, often challenging existing pre-conceptions about structure and function of biological systems. For tissue and cell structure imaging, the move from 2D section-based microscopy to true 3D data collection has been a major driver of new insight. In the sub-cellular domain, electron tomography is a powerful technique for exploration of cellular structures in 3D with unparalleled fidelity at nanometer resolution.Electron tomography is particularly advantageous for studying highly compartmentalised cells such as cardiomyocytes, where elaborate sub-cellular structures play crucial roles in electrophysiology and mechanics. Although the anatomy of specific ultra-structures, such as dyadic couplons, has been extensively explored using 2D electron microscopy of thin sections, we still lack accurate, quantitative knowledge of true individual shape, volume and surface area of sub-cellular domains, as well as their 3D spatial interrelations; let alone of how these are reshaped during the cycle of contraction and relaxation. Here we discuss and illustrate the utility of ET for identification, visualisation, and analysis of 3D cardiomyocyte ultrastructures such as the T-tubular system, sarcoplasmic reticulum, mitochondria and microtubules.

Johnston CM, Han JC, Ruddy BP, Nielsen PM, Taberner AJ. A high-resolution thermoelectric module-based calorimeter for measuring the energetics of isolated ventricular trabeculae at body temperature. Am J Physiol Heart Circ Physiol 309: H318 -H324, 2015. First published May 22, 2015; doi:10.1152/ajpheart.00194.2015.-Isolated ventricular trabeculae are the most common experimental preparations used in the study of cardiac energetics. However, the experiments have been conducted at subphysiological temperatures. We have overcome this limitation by designing and constructing a novel calorimeter with sufficiently high thermal resolution for simultaneously measuring the heat output and force production of isolated, contracting, ventricular trabeculae at body temperature. This development was largely motivated by the need to better understand cardiac energetics by performing such measurements at body temperature to relate tissue performance to whole heart behavior in vivo. Our approach uses solid-state thermoelectric modules, tailored for both temperature sensing and temperature control. The thermoelectric modules have high sensitivity and low noise, which, when coupled with a multilevel temperature control system, enable an exceptionally high temperature resolution with a noise-equivalent power an order of magnitude greater than those of other existing muscle calorimeters. Our system allows us to rapidly and easily change the experimental temperature without disturbing the state of the muscle. Our calorimeter is useful in many experiments that explore the energetics of normal physiology as well as pathophysiology of cardiac muscle. muscle energetics; microcalorimetry; heat production NEW & NOTEWORTHY We have developed a new muscle calorimeter, which has enabled the first simultaneous measurements of the force and heat output of isolated, actively contracting, cardiac trabeculae at body temperature. This was achieved by the use of thermoelectric modules for high-resolution temperature sensing and precise temperature control.MEASUREMENTS OF THE ENERGETICS of cardiac muscle under controlled experimental conditions, and in particular at body temperature, enable better understanding of cardiac muscle function in health and disease. Studies of force production simultaneous with heat output of cardiac muscle require the use of isolated tissue preparations such as small papillary muscles (2-4, 12, 26, 34) or trabeculae carneae (17-21) as they are sufficiently small to avoid muscle anoxia (14). The use of these tissue preparations, which have approximately one-dimensional arrangements of myocytes, simplifies the interpretation of measurements. Although these tissue studies have improved our understanding of myocardial energetics, previous experiments have been performed only at subphysiological temperatures, typically 10°C below that of body temperature (3,5,10,11,13,23,25,26,28,30,31). One exception is the study by Widén (39), who made simultaneous force and heat measurements in mouse papillary muscles at body temperature. H...

Long‐term systemic arterial hypertension, and its associated compensatory response of left‐ventricular hypertrophy, is fatal. This disease leads to cardiac failure and culminates in death. The spontaneously hypertensive rat (SHR) is an excellent animal model for studying this pathology, suffering from ventricular failure beginning at about 18 months of age. In this study, we isolated left‐ventricular trabeculae from SHR‐F hearts and contrasted their mechanoenergetic performance with those from nonfailing SHR (SHR‐NF) and normotensive Wistar rats. Our results show that, whereas the performance of the SHR‐F differed little from that of the SHR‐NF, both SHR groups performed less stress‐length work than that of Wistar trabeculae. Their lower work output arose from reduced ability to produce sufficient force and shortening. Neither their heat production nor their enthalpy output (the sum of work and heat), particularly the energy cost of Ca2+ cycling, differed from that of the Wistar controls. Consequently, mechanical efficiency (the ratio of work to change of enthalpy) of both SHR groups was lower than that of the Wistar trabeculae. Our data suggest that in hypertension‐induced left‐ventricular hypertrophy, the mechanical performance of the tissue is compromised such that myocardial efficiency is reduced.

Abstract:In optogenetics, light-activated proteins are used to monitor and modulate cellular behaviour with light. Combining genetic targeting of distinct cellular populations with defined patterns of optical stimulation enables one to study specific cell classes in complex biological tissues. In the current study we attempted to investigate the functional relevance of heterocellular electrotonic coupling in cardiac tissue in situ. In order to do that, we used a Cre-Lox approach to express the light-gated cation channel Channelrhodopsin-2 (ChR2) specifically in either cardiac myocytes or non-myocytes. Despite high specificity when using the same Cre driver lines in a previous study in combination with a different optogenetic probe, we found patchy off-target ChR2 expression in cryo-sections and extended z-stack imaging through the ventricular wall of hearts cleared using CLARITY. Based on immunohistochemical analysis, single-cell electrophysiological recordings and whole-genome sequencing, we reason that non-specificity is caused on the Cre recombination level. Our study highlights the importance of careful design and validation of the Cre recombination targets for reliable cell class specific expression of optogenetic tools.Introduction: