The mechanism of cardiac resynchronization therapy (
CRT
) remains unclear. In this study, mitochondria calcium uniporter (
MCU
), dynamin‐related protein‐1 (
DNM
1L/Drp1) and their relationship with autophagy in heart failure (
HF
) and
CRT
are investigated. Thirteen male beagle's dogs were divided into three groups (sham,
HF
,
CRT
). Animals received left bundle branch (
LBB
) ablation followed by either 8‐week rapid atrial pacing or 4‐week rapid atrial pacing and 4‐week biventricular pacing. Cardiac function was evaluated by echocardiography. Differentially expressed genes (
DEG
s) were detected by microarray analysis. General morphological changes, mitochondrial ultrastructure, autophagosomes and mitophagosomes were investigated. The cardiomyocyte stretching was adopted to imitate the mechanical effect of
CRT
. Cells were divided into three groups (control, angiotensin‐
II
and angiotensin‐
II
+ stretching).
MCU
,
DNM
1L/Drp1 and autophagy markers were detected by western blots or immunofluorescence. In the present study,
CRT
could correct cardiac dysfunction, decrease cardiomyocyte's size, alleviate cardiac fibrosis, promote the formation of autophagosome and mitigate mitochondrial injury.
CRT
significantly influenced gene expression profile, especially down‐regulating
MCU
and up‐regulating
DNM
1L/Drp1. Cell stretching reversed the angiotensin‐
II
induced changes of
MCU
and
DNM
1L/Drp1 and partly restored autophagy.
CRT
's mechanical effects down‐regulated
MCU
, up‐regulated
DNM
1L/Drp1 and subsequently enhanced autophagy. Besides, the mechanical stretching prevented the angiotensin‐
II
‐induced cellular enlargement.