Background Cardiac recovery in response to mechanical unloading by left ventricular assist devices (LVADs) has been demonstrated in subgroups of chronic heart failure (HF) patients. Hallmarks of HF are depletion and disorganization of the transverse tubular system (t-system) in cardiomyocytes. Here, we investigated remodeling of the t-system in human end-stage HF and its role in cardiac recovery. Methods Left ventricular biopsies were obtained from 5 donors (CTRL) and 26 chronic HF patients undergoing implantation of LVADs. Three-dimensional confocal microscopy and computational image analysis were applied to assess t-system structure, density, and distance of ryanodine receptor (RyR) clusters to the sarcolemma, including the t-system. Recovery of cardiac function in response to mechanical unloading was assessed by echocardiography during turn-down of the LVAD. Results The majority of HF myocytes showed remarkable t-system remodeling, particularly sheet-like invaginations of the sarcolemma. Circularity of t-system components was decreased in HF vs CTRL (0.37±0.01 vs 0.46±0.02, p<0.01), and the volume/length ratio was increased in HF (0.36±0.01μm2 vs 0.25±0.02μm2, p<0.0001). T-system density was reduced in HF, leading to increased RyR-sarcolemma distances (0.96±0.05μm vs 0.64±0.1μm, p<0.01). Low RyR-sarcolemma distances at time of LVAD implantation predicted high post-LVAD left-ventricular ejection fractions (EF, p<0.01) and EF increase during unloading (p<0.01). EF in patients with pre-LVAD RyR-sarcolemma distances larger than 1μm did not improve following mechanical unloading. Additionally, calcium transients were recorded in field-stimulated isolated human cardiomyocytes and analyzed with respect to local t-system density. Calcium release in HF myocytes was restricted to regions proximal to the sarcolemma. Local calcium upstroke was delayed (23.9±4.9ms vs 10.3±1.7ms, p<0.05) and more asynchronous (18.1ms±1.5ms vs 8.9±2.2ms, p<0.01) in HF cells with low t-system density versus cells with high t-system density. Conclusions The t-system in end-stage human HF presents a characteristic novel phenotype consisting of sheet-like invaginations of the sarcolemma. Our results suggest that the remodeled t-system impairs excitation-contraction coupling and functional recovery during chronic LVAD unloading. An intact t-system at time of LVAD implantation may constitute a precondition and predictor for functional cardiac recovery following mechanical unloading.

Transient receptor potential canonical 1 (TRPC1) protein is abundantly expressed in cardiomyocytes. While TRPC1 is supposed to be critically involved in cardiac hypertrophy, its physiological role in cardiomyocytes is poorly understood. We investigated the subcellular location of TRPC1 and its contribution to Ca 2+ signaling in mammalian ventricular myocytes. Immunolabeling, three-dimensional scanning confocal microscopy and quantitative colocalization analysis revealed an abundant intracellular location of TRPC1 in neonatal rat ventricular myocytes (NRVMs) and adult rabbit ventricular myocytes. TRPC1 was colocalized with intracellular proteins including sarco/endoplasmic reticulum Ca 2+ ATPase 2 in the sarcoplasmic reticulum (SR). Colocalization with wheat germ agglutinin, which labels the glycocalyx and thus marks the sarcolemma including the transverse tubular system, was low. Super-resolution and immunoelectron microscopy supported the intracellular location of TRPC1. We investigated Ca 2+ signaling in NRVMs after adenoviral TRPC1 overexpression or silencing. In NRVMs bathed in Na + and Ca 2+ free solution, TRPC1 overexpression and silencing was associated with a decreased and increased SR Ca 2+ content, respectively. In isolated rabbit cardiomyocytes bathed in Na + and Ca 2+ free solution, we found an increased decay of the cytosolic Ca 2+ concentration [Ca 2+ ] i and increased SR Ca 2+ content in the presence of the TRPC channel blocker SKF-96365. In a computational model of rabbit ventricular myocytes at physiological pacing rates, Ca 2+ leak through SR TRPC channels increased the systolic and diastolic [Ca 2+ ] i with only minor effects on the action potential and SR Ca 2+ content. Our studies suggest that TRPC1 channels are localized in the SR, and not present in the sarcolemma of ventricular myocytes. The studies provide evidence for a role of TRPC1 as a contributor to SR Ca 2+ leak in cardiomyocytes, which was previously explained by ryanodine receptors only. We propose that the findings will guide us to an understanding of TRPC1 channels as modulators of [Ca 2+ ] i and contractility in cardiomyocytes.

No abstract

Transient receptor potential canonical 1 (TRPC1) channels are Ca2+-permeable ion channels expressed in cardiomyocytes. An involvement of TRPC1 channels in cardiac diseases is widely established. However, the physiological role of TRPC1 channels and the mechanisms through which they contribute to disease development are still under investigation. Our prior work suggested that TRPC1 forms Ca2+ leak channels located in the sarcoplasmic reticulum (SR) membrane. Prior studies suggested that TRPC1 channels in the cell membrane are mechanosensitive, but this was not yet investigated in cardiomyocytes or for SR localized TRPC1 channels. We applied adenoviral transfection to overexpress or suppress TRPC1 expression in neonatal rat ventricular myocytes (NRVMs). Transfections were evaluated with RT-qPCR, western blot, and fluorescent imaging. Single-molecule localization microscopy revealed high colocalization of exogenously expressed TRPC1 and the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2). To test our hypothesis that TRPC1 channels contribute to mechanosensitive Ca2+ SR leak, we directly measured SR Ca2+ concentration ([Ca2+]SR) using adenoviral transfection with a novel ratiometric genetically encoded SR-targeting Ca2+ sensor. We performed fluorescence imaging to quantitatively assess [Ca2+]SR and leak through TRPC1 channels of NRVMs cultured on stretchable silicone membranes. [Ca2+]SR was increased in cells with suppressed TRPC1 expression vs. control and Transient receptor potential canonical 1-overexpressing cells. We also detected a significant reduction in [Ca2+]SR in cells with Transient receptor potential canonical 1 overexpression when 10% uniaxial stretch was applied. These findings indicate that TRPC1 channels underlie the mechanosensitive modulation of [Ca2+]SR. Our findings are critical for understanding the physiological role of TRPC1 channels and support the development of pharmacological therapies for cardiac diseases.

Transient receptor potential canonical 6 (TRPC6) channels are non-selective cation channels that are thought to underlie mechano-modulation of calcium signaling in cardiomyocytes. TRPC6 channels are involved in development of cardiac hypertrophy and related calcineurin-nuclear factor of activated T cells (NFAT) signaling. However, the exact location and roles of TRPC6 channels remain ill-defined in cardiomyocytes. We used an expression system based on neonatal rat ventricular myocytes (NRVMs) to investigate the location of TRPC6 channels and their role in calcium signaling. NRVMs isolated from 1-to 2-day-old animals were cultured and infected with an adenoviral vector to express enhanced-green fluorescent protein (eGFP) or TRPC6-eGFP. After 3 days, NRVMs were fixed, immunolabeled, and imaged with confocal and super-resolution microscopy to determine TRPC6 localization. Cytosolic calcium transients at 0.5 and 1 Hz pacing rates were recorded in NRVMs using indo-1, a ratio-metric calcium dye. Confocal and superresolution microscopy suggested that TRPC6-eGFP localized to the sarcolemma. NRVMs infected with TRPC6-eGFP exhibited higher diastolic and systolic cytosolic calcium concentration as well as increased sarcoplasmic reticulum (SR) calcium load compared to eGFP infected cells. We applied a computer model comprising sarcolemmal TRPC6 current to explain our experimental findings. Altogether, our studies indicate that TRPC6 channels play a role in sarcolemmal and intracellular calcium signaling in cardiomyocytes. Our findings support the hypothesis that upregulation or activation of TRPC6 channels, e.g., in disease, leads to sustained elevation of the cytosolic calcium concentration, which is thought to activate calcineurin-NFAT signaling and cardiac hypertrophic remodeling. Also, our findings support the hypothesis that mechanosensitivity of TRPC6 channels modulates cytosolic calcium transients and SR calcium load.